Microorganisms and methods for production of 4-hydroxybutyrate, 1,4-butanediol and related compounds

ABSTRACT

The invention provides non-naturally occurring microbial organisms having a 4-hydroxybutyrate pathway and being capable of producing 4-hydroxybutyrate, wherein the microbial organism comprises one or more genetic modifications. The invention additionally provides methods of producing 4-hydroxybutyrate or related products using the microbial organisms.

This application claims the benefit of priority of U.S. ProvisionalApplication Ser. No. 61/655,429, filed Jun. 4, 2012, the entire contentsof which are incorporated herein by reference.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 29, 2013, isnamed 12956-191-999_SeqList.txt and is 238,427 bytes in size.

This invention relates generally to in silico design of organisms andengineering of organisms, more particularly to organisms having1,4-butanediol, 4-hydroxybutyryl-CoA, 4-hydroxybutanal or putrescinebiosynthesis capability.

BACKGROUND OF THE INVENTION

The compound 4-hydroxybutanoic acid (4-hydroxybutanoate,4-hydroxybutyrate, 4-HB) is a 4-carbon carboxylic acid that hasindustrial potential as a building block for various commodity andspecialty chemicals. In particular, 4-HB has the potential to serve as anew entry point into the 1,4-butanediol family of chemicals, whichincludes solvents, resins, polymer precursors, and specialty chemicals.1,4-Butanediol (BDO) is a polymer intermediate and industrial solventwith a global market of about 3 billion lb/year. BDO is currentlyproduced from petrochemical precursors, primarily acetylene, maleicanhydride, and propylene oxide.

For example, acetylene is reacted with 2 molecules of formaldehyde inthe Reppe synthesis reaction (Kroschwitz and Grant, Encyclopedia ofChem. Tech., John Wiley and Sons, Inc., New York (1999)), followed bycatalytic hydrogenation to form 1,4-butanediol. It has been estimatedthat 90% of the acetylene produced in the U.S. is consumed forbutanediol production. Alternatively, it can be formed by esterificationand catalytic hydrogenation of maleic anhydride, which is derived frombutane. Downstream, butanediol can be further transformed; for example,by oxidation to γ-butyrolactone, which can be further converted topyrrolidone and N-methyl-pyrrolidone, or hydrogenolysis totetrahydrofuran. These compounds have varied uses as polymerintermediates, solvents, and additives, and have a combined market ofnearly 2 billion lb/year. It is desirable to develop a method forproduction of these chemicals by alternative means that not onlysubstitute renewable for petroleum-based feedstocks, and also use lessenergy- and capital-intensive processes.

Thus, there exists a need for alternative means for effectivelyproducing commercial quantities of 1,4-butanediol and its chemicalprecursors. The present invention satisfies this need and providesrelated advantages as well.

SUMMARY OF INVENTION

The invention provides non-naturally occurring microbial organismshaving a 4-hydroxybutyrate pathway and being capable of producing4-hydroxybutyrate, wherein the microbial organism comprises one or moregenetic modifications. The invention additionally provides methods ofproducing 4-hydroxybutyrate or related products using the microbialorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing biochemical pathways to4-hydroxybutyurate (4-HB) and to 1,4-butanediol production. The first 5steps are endogenous to E. coli, while the remainder can be expressedheterologously. Enzymes catalyzing the biosynthetic reactions are: (1)succinyl-CoA synthetase; (2) CoA-independent succinic semialdehydedehydrogenase; (3) α-ketoglutarate dehydrogenase; (4) glutamate:succinate semialdehyde transaminase; (5) glutamate decarboxylase; (6)CoA-dependent succinic semialdehyde dehydrogenase; (7)4-hydroxybutanoate dehydrogenase; (8) α-ketoglutarate decarboxylase; (9)4-hydroxybutyryl CoA:acetyl-CoA transferase; (10) butyrate kinase; (11)phosphotransbutyrylase; (12) aldehyde dehydrogenase; (13) alcoholdehydrogenase.

FIG. 2 is a schematic diagram showing homoserine biosynthesis in E.coli.

FIGS. 3(a)-3(c) show the production of 4-HB in glucose minimal mediumusing E. coli strains harboring plasmids expressing various combinationsof 4-HB pathway genes. (FIG. 3a ) 4-HB concentration in culture broth;(FIG. 3b ) succinate concentration in culture broth; (FIG. 3c ) cultureOD, measured at 600 nm. Clusters of bars represent the 24 hour, 48 hour,and 72 hour (if measured) timepoints. The codes along the x-axisindicate the strain/plasmid combination used. The first index refers tothe host strain: 1, MG1655 lacI^(Q); 2, MG1655 ΔgabD lacI^(Q); 3, MG1655ΔgabD ΔaldA lacI^(Q). The second index refers to the plasmid combinationused: 1, pZE13-0004-0035 and pZA33-0036; 2, pZE13-0004-0035 andpZA33-0010n; 3, pZE13-0004-0008 and pZA33-0036; 4, pZE13-0004-0008 andpZA33-0010n; 5, Control vectors pZE13 and pZA33.

FIG. 4 shows the production of 4-HB from glucose in E. coli strainsexpressing α-ketoglutarate decarboxylase from Mycobacteriumtuberculosis. Strains 1-3 contain pZE13-0032 and pZA33-0036. Strain 4expresses only the empty vectors pZE13 and pZA33. Host strains are asfollows: 1 and 4, MG1655 lacI^(Q); 2, MG1655 ΔgabD lacI^(Q); 3, MG1655ΔgabD ΔaldA lacI^(Q). The bars refer to concentration at 24 and 48hours.

FIG. 5 shows the production of BDO from 10 mM 4-HB in recombinant E.coli strains. Numbered positions correspond to experiments with MG1655lacI^(Q) containing pZA33-0024, expressing cat2 from P. gingivalis, andthe following genes expressed on pZE13: 1, none (control); 2, 0002; 3,0003; 4, 0003n; 5, 0011; 6, 0013; 7, 0023; 8, 0025; 9, 0008n; 10, 0035.Gene numbers are defined in Table 6. For each position, the bars referto aerobic, microaerobic, and anaerobic conditions, respectively.Microaerobic conditions were created by sealing the culture tubes butnot evacuating them.

FIGS. 6(a)-6(h) show the mass spectrum of 4-HB and BDO produced byMG1655 lacI^(Q) pZE13-0004-0035-0002 pZA33-0034-0036 grown in M9 minimalmedium supplemented with 4 g/L unlabeled glucose (FIGS. 6(a), 6(c),6(e), and 6(g)) uniformly labeled ¹³C-glucose (FIGS. 6(b), 6(d), 6(f),and 6(h)). 6(a) and 6(b), mass 116 characteristic fragment ofderivatized BDO, containing 2 carbon atoms; 6(c) and 6(d), mass 177characteristic fragment of derivatized BDO, containing 1 carbon atom;6(e) and 6(f), mass 117 characteristic fragment of derivatized 4-HB,containing 2 carbon atoms; 6(g) and 6(h), mass 233 characteristicfragment of derivatized 4-HB, containing 4 carbon atoms.

FIG. 7 is a schematic process flow diagram of bioprocesses for theproduction of γ-butyrolactone. Panel (a) illustrates fed-batchfermentation with batch separation and panel (b) illustrates fed-batchfermentation with continuous separation.

FIGS. 8A and 8B show exemplary 1,4-butanediol (BDO) pathways. FIG. 8Ashows BDO pathways from succinyl-CoA. FIG. 8B shows BDO pathways fromalpha-ketoglutarate.

FIGS. 9A-9C show exemplary BDO pathways. FIGS. 9A and 9B show pathwaysfrom 4-aminobutyrate. FIG. 9C shows a pathway from acetoactyl-CoA to4-aminobutyrate.

FIG. 10 shows exemplary BDO pathways from alpha-ketoglutarate.

FIG. 11 shows exemplary BDO pathways from glutamate.

FIG. 12 shows exemplary BDO pathways from acetoacetyl-CoA.

FIG. 13 shows exemplary BDO pathways from homoserine.

FIGS. 14A-14C show the nucleotide and amino acid sequences of E. colisuccinyl-CoA synthetase. FIG. 14A shows the nucleotide sequence (SEQ IDNO:46) of the E. coli sucCD operon. FIGS. 14B (SEQ ID NO:47) and 14C(SEQ ID NO:48) show the amino acid sequences of the succinyl-CoAsynthetase subunits encoded by the sucCD operon.

FIGS. 15A and 15B show the nucleotide and amino acid sequences ofMycobacterium bovis alpha-ketoglutarate decarboxylase. FIG. 15A showsthe nucleotide sequence (SEQ ID NO:49) of Mycobacterium bovis sucA gene.FIG. 15B shows the amino acid sequence (SEQ ID NO:50) of M. bovisalpha-ketoglutarate decarboxylase.

FIG. 16 shows biosynthesis in E. coli of 4-hydroxybutyrate from glucosein minimal medium via alpha-ketoglutarate under anaerobic (microaerobic)conditions. The host strain is ECKh-401. The experiments are labeledbased on the upstream pathway genes present on the plasmid pZA33 asfollows: 1) 4hbd-sucA; 2) sucCD-sucD-4-hbd; 3) sucCD-sucD-4-hbd-sucA.

FIG. 17 shows biosynthesis in E. coli of 4-hydroxybutyrate from glucosein minimal medium via succinate and alpha-ketoglutarate. The host strainis wild-type MG1655. The experiments are labeled based on the genespresent on the plasmids pZE13 and pZA33 as follows: 1) empty controlvectors 2) empty pZE13, pZA33-4-hbd; 3) pZE13-sucA, pZA33-4-hbd.

FIG. 18 A shows the nucleotide sequence (SEQ ID NO:51) of CoA-dependentsuccinate semialdehyde dehydrogenase (sucD) from Porphyromonasgingivalis, and FIG. 18B shows the encoded amino acid sequence (SEQ IDNO:52).

FIG. 19A shows the nucleotide sequence (SEQ ID NO:53) of4-hydroxybutyrate dehydrogenase (4hbd) from Porphymonas gingivalis, andFIG. 19B shows the encoded amino acid sequence (SEQ ID NO:54).

FIG. 20A shows the nucleotide sequence (SEQ ID NO:55) of4-hydroxybutyrate CoA transferase (cat2) from Porphyromonas gingivalis,and FIG. 20B shows the encoded amino acid sequence (SEQ ID NO:56).

FIG. 21A shows the nucleotide sequence (SEQ ID NO:57) ofphosphotransbutyrylase (ptb) from Clostridium acetobutylicum, and FIG.21B shows the encoded amino acid sequence (SEQ ID NO:58).

FIG. 22A shows the nucleotide sequence (SEQ ID NO:59) of butyrate kinase(buk1) from Clostridium acetobutylicum, and FIG. 22B shows the encodedamino acid sequence (SEQ ID NO:60).

FIGS. 23A-23D show alternative nucleotide sequences for C.acetobutylicum 020 (phosphtransbutyrylase) with altered codons for moreprevalent E. coli codons relative to the C. acetobutylicum nativesequence. FIGS. 23A-23D (020A-020D, SEQ ID NOS:61-64, respectively)contain sequences with increasing numbers of rare E. coli codonsreplaced by more prevalent codons (A<B<C<D).

FIGS. 24A-24D show alternative nucleotide sequences for C.acetobuytlicum 021 (butyrate kinase) with altered codons for moreprevalent E. coli codons relative to the C. acetobutylicum nativesequence. FIGS. 24A-24D (021A-021B, SEQ ID NOS:65-68, respectively)contain sequences with increasing numbers of rare E. coli codonsreplaced by more prevalent codons (A<B<C<D).

FIGS. 25A and 25B show improved expression of butyrate kinase (BK) andphosphotransbutyrylase (PTB) with optimized codons for expression in E.coli. FIG. 25A shows sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) stained for proteins with Coomassie blue;lane 1, control vector with no insert; lane 2, expression of C.acetobutylicum native sequences in E. coli; lane 3, expression of020B-021B codon optimized PTB-BK; lane 4, expression of 020C-021C codonoptimized PTB-BK. The positions of BK and PTB are shown. FIG. 25B showsthe BK and PTB activities of native C. acetobutylicum sequence (2021n)compared to codon optimized 020B-021B (2021B) and 020C-021C (2021C).

FIG. 26 shows production of BDO and gamma-butyrylactone (GBL) in variousstrains expressing BDO producing enzymes: Cat2 (034); 2021n; 2021B;2021C.

FIG. 27A shows the nucleotide sequence (SEQ ID NO:69) of the nativeClostridium biejerinckii ald gene (025n), and FIG. 27B shows the encodedamino acid sequence (SEQ ID NO:70).

FIGS. 28A-28D show alternative gene sequences for the Clostridiumbeijerinckii ald gene (025A-025D, SEQ ID NOS:71-74, respectively), inwhich increasing numbers of rare codons are replaced by more prevalentcodons (A<B<C<D).

FIG. 29 shows expression of native C. beijerinckii ald gene and codonoptimized variants; no ins (control with no insert), 025n, 025A, 025B,025C, 025D.

FIGS. 30A and 30B show BDO or BDO and ethanol production in variousstrains.

FIG. 30A shows BDO production in strains containing the native C.beijerinckii ald gene (025n) or variants with optimized codons forexpression in E. coli (025A-025D). FIG. 30B shows production of ethanoland BDO in strains expressing the C. acetobutylicum AdhE2 enzyme (002C)compared to the codon optimized variant 025B. The third set showsexpression of P. gingivalis sucD (035). In all cases, P. gingivalis Cat2(034) is also expressed.

FIG. 31A shows the nucleotide sequence (SEQ ID NO:75) of the adh1 genefrom Geobacillus thermoglucosidasius, and FIG. 31B shows the encodedamino acid sequence (SEQ ID NO:76).

FIG. 32A shows the expression of the Geobacillus thermoglucosidasiusadh1 gene in E. coli. Either whole cell lysates or supernatants wereanalyzed by SDS-PAGE and stained with Coomassie blue for plasmid with noinsert, plasmid with 083 (Geotrichum capitatum N-benzyl-3-pyrrolidinoldehydrogenase) and plasmid with 084 (Geobacillus thermoglucosidasiusadh1) inserts. FIG. 32B shows the activity of 084 with butyraldehyde(diamonds) or 4-hydroxybutyraldehyde (squares) as substrates.

FIG. 33 shows the production of BDO in various strains: plasmid with noinsert; 025B, 025B-026n; 025B-026A; 025B-026B; 025B-026C; 025B-050;025B-052; 025B-053; 025B-055; 025B-057; 025B-058; 025B-071; 025B-083;025B-084; PTSlacO-025B; PTSlacO-025B-026n.

FIG. 34 shows a plasmid map for the vector pRE118-V2.

FIG. 35 shows the sequence (SEQ ID NO:77) of the ECKh-138 regionencompassing the aceF and lpdA genes. The K. pneumonia lpdA gene isunderlined, and the codon changed in the Glu354Lys mutant shaded.

FIG. 36 shows the protein sequence comparison of the native E. coli lpdA(SEQ ID NO:78) and the mutant K. pneumonia lpdA (SEQ ID NO:79).

FIG. 37 shows 4-hydroxybutyrate (left bars) and BDO (right bars)production in the strains AB3, MG1655 ΔldhA and ECKh-138. All strainsexpressed E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd on themedium copy plasmid pZA33, and P. gingivalis Cat2, C. acetobutylicumAdhE2 on the high copy plasmid pZE13.

FIG. 38 shows the nucleotide sequence (SEQ ID NO:80) of the 5′ end ofthe aceE gene fused to the pflB-p6 promoter and ribosome binding site(RBS). The 5′ italicized sequence shows the start of the aroP gene,which is transcribed in the opposite direction from the pdh operon. The3′ italicized sequence shows the start of the aceE gene. In upper case:pflB RBS. Underlined: FNR binding site. In bold: pflB-p6 promotersequence.

FIG. 39 shows the nucleotide sequence (SEQ ID NO:81) in the aceF-lpdAregion in the strain ECKh-456.

FIG. 40 shows the production of 4-hydroxybutyrate, BDO and pyruvate(left to right bars, respectively) for each of strains ECKh-439,ECKh-455 and ECKh-456.

FIG. 41A shows a schematic of the recombination sites for deletion ofthe mdh gene. FIG. 41B shows the sequence (nucleotide sequence SEQ IDNO:82; amino acid sequence SEQ ID NO:83) of the PCR product of theamplification of chloramphenicol resistance gene (CAT) flanked by FRTsites and homology regions from the mdh gene from the plasmid pKD3.

FIG. 42 shows the sequence (SEQ ID NO:84) of the arcA deleted region instrain ECKh-401.

FIG. 43 shows the sequence (SEQ ID NO:85) of the region encompassing amutated gltA gene of strain ECKh-422.

FIGS. 44A and 44B show the citrate synthase activity of wild type gltAgene product and the R163L mutant. The assay was performed in theabsence (diamonds) or presence of 0.4 mM NADH (squares).

FIG. 45 shows the 4-hydroxybutyrate (left bars) and BDO (right bars)production in strains ECKh-401 and ECKh-422, both expressing genes forthe complete BDO pathway on plasmids.

FIG. 46 shows central metabolic fluxes and associated 95% confidenceintervals from metabolic labeling experiments. Values are molar fluxesnormalized to a glucose uptake rate of 1 mmol/hr. The result indicatesthat carbon flux is routed through citrate synthase in the oxidativedirection and that most of the carbon enters the BDO pathway rather thancompleting the TCA cycle.

FIGS. 47A and 47B show extracellular product formation for strainsECKh-138 and ECKh-422, both expressing the entire BDO pathway onplasmids. The products measured were acetate (Ace), pyruvate (Pyr),4-hydroxybutyrate (4HB), 1,4-butanediol (BDO), ethanol (EtOH), and otherproducts, which include gamma-butyrolactone (GBL), succinate, andlactate.

FIG. 48 shows the sequence (SEQ ID NO:86) of the region followingreplacement of PEP carboxylase (ppc) by H. influenzaephosphoenolpyruvate carboxykinase (pepck). The pepck coding region isunderlined.

FIG. 49 shows growth of evolved pepCK strains grown in minimal mediumcontaining 50 mM NaHCO₃.

FIG. 50 shows product formation in strain ECKh-453 expressing P.gingivalis Cat2 and C. beijerinckii Ald on the plasmid pZS*13. Theproducts measured were 1,4-butanediol (BDO), pyruvate, 4-hydroxybutyrate(4HB), acetate, γ-butyrolactone (GBL) and ethanol.

FIG. 51 shows BDO production of two strains, ECKh-453 and ECKh-432. Bothcontain the plasmid pZS*13 expressing P. gingivalis Cat2 and C.beijerinckii Ald. The cultures were grown under microaerobic conditions,with the vessels punctured with 27 or 18 gauge needles, as indicated.

FIG. 52 shows the nucleotide sequence (SEQ ID NO:87) of the genomic DNAof strain ECKh-426 in the region of insertion of a polycistronic DNAfragment containing a promoter, sucCD gene, sucD gene, 4hbd gene and aterminator sequence.

FIG. 53 shows the nucleotide sequence (SEQ ID NO:88) of the chromosomalregion of strain ECKh-432 in the region of insertion of a polycistronicsequence containing a promoter, sucA gene, C. kluyveri 4hbd gene and aterminator sequence.

FIG. 54 shows BDO synthesis from glucose in minimal medium in theECKh-432 strain having upstream BDO pathway encoding genes intregratedinto the chromosome and containing a plasmid harboring downstream BDOpathway genes.

FIG. 55 shows a PCR product (SEQ ID NO:89) containing thenon-phosphotransferase (non-PTS) sucrose utilization genes flanked byregions of homology to the rrnC region.

FIG. 56 shows a schematic diagram of the integrations site in the rrnCoperon.

FIG. 57 shows average product concentration, normalized to cultureOD600, after 48 hours of growth of strain ECKh-432 grown on glucose andstrain ECKh-463 grown on sucrose. Both contain the plasmid pZS*13expressing P. gingivalis Cat2 and C. beijerinckii Ald. The data is for 6replicate cultures of each strain. The products measured were1,4-butanediol (BDO), 4-hydroxybutyrate (4HB), γ-butyrolactone (GBL),pyruvate (PYR) and acetate (ACE) (left to right bars, respectively).

FIG. 58 shows exemplary pathways to 1,4-butanediol from succcinyl-CoAand alpha-ketoglutarate. Abbreviations: A) Succinyl-CoA reductase(aldehyde forming), B) Alpha-ketoglutarate decarboxylase, C)4-Hydroxybutyrate dehydrogenase, D) 4-Hydroxybutyrate reductase, E)1,4-Butanediol dehydrogenase.

FIG. 59A shows the nucleotide sequence (SEQ ID NO:90) of carboxylic acidreductase from Nocardia iowensis (GNM_720), and FIG. 59B shows theencoded amino acid sequence (SEQ ID NO:91).

FIG. 60A shows the nucleotide sequence (SEQ ID NO:92) ofphosphpantetheine transferase, which was codon optimized, and FIG. 60Bshows the encoded amino acid sequence (SEQ ID NO:93).

FIG. 61 shows a plasmid map of plasmid pZS*-13S-720 72lopt.

FIGS. 62A and 62B show pathways to 1,4-butanediol from succinate,succcinyl-CoA, and alpha-ketoglutarate. Abbreviations: A) Succinyl-CoAreductase (aldehyde forming), B) Alpha-ketoglutarate decarboxylase, C)4-Hydroxybutyrate dehydrogenase, D) 4-Hydroxybutyrate reductase, E)1,4-Butanediol dehydrogenase, F) Succinate reductase, G) Succinyl-CoAtransferase, H) Succinyl-CoA hydrolase, I) Succinyl-CoA synthetase (orSuccinyl-CoA ligase), J) Glutamate dehydrogenase, K) Glutamatetransaminase, L) Glutamate decarboxylase, M) 4-aminobutyratedehydrogenase, N) 4-aminobutyrate transaminase, O) 4-Hydroxybutyratekinase, P) Phosphotrans-4-hydroxybutyrylase, Q) 4-Hydroxybutyryl-CoAreductase (aldehyde forming), R) 4-hydroxybutyryl-phosphate reductase,S) Succinyl-CoA reductase (alcohol forming), T) 4-Hydroxybutyryl-CoAtransferase, U) 4-Hydroxybutyryl-CoA hydrolase, V) 4-Hydroxybutyryl-CoAsynthetase (or 4-Hydroxybutyryl-CoA ligase), W) 4-Hydroxybutyryl-CoAreductase (alcohol forming), X) Alpha-ketoglutarate reductase, Y)5-Hydroxy-2-oxopentanoate dehydrogenase, Z) 5-Hydroxy-2-oxopentanoatedecarboxylase, AA) 5-hydroxy-2-oxopentanoate dehydrogenase(decarboxylation).

FIG. 63 shows pathways to putrescine from succinate, succcinyl-CoA, andalpha-ketoglutarate. Abbreviations: A) Succinyl-CoA reductase (aldehydeforming), B) Alpha-ketoglutarate decarboxylase, C) 4-Aminobutyratereductase, D) Putrescine dehydrogenase, E) Putrescine transaminase, F)Succinate reductase, G) Succinyl-CoA transferase, H) Succinyl-CoAhydrolase, I) Succinyl-CoA synthetase (or Succinyl-CoA ligase), J)Glutamate dehydrogenase, K) Glutamate transaminase, L) Glutamatedecarboxylase, M) 4-Aminobutyrate dehydrogenase, N) 4-Aminobutyratetransaminase, O) Alpha-ketoglutarate reductase, P)5-Amino-2-oxopentanoate dehydrogenase, Q) 5-Amino-2-oxopentanoatetransaminase, R) 5-Amino-2-oxopentanoate decarboxylase, S) Ornithinedehydrogenase, T) Ornithine transaminase, U) Ornithine decarboxylase.

FIG. 64A shows the nucleotide sequence (SEQ ID NO:94) of carboxylic acidreductase from Mycobacterium smegmatis mc(2)155 (designated 890), andFIG. 64B shows the encoded amino acid sequence (SEQ ID NO:95).

FIG. 65A shows the nucleotide sequence (SEQ ID NO:96) of carboxylic acidreductase from Mycobacterium avium subspecies paratuberculosis K-10(designated 891), and FIG. 65B shows the encoded amino acid sequence(SEQ ID NO:97).

FIG. 66A shows the nucleotide sequence (SEQ ID NO:98) of carboxylic acidreductase from Mycobacterium marinum M (designated 892), and FIG. 66Bshows the encoded amino acid sequence (SEQ ID NO:99).

FIG. 67 shows a schematic of the E. coli MG1655 chromosome betweennucleotides 760928 and 765930.

FIGS. 68A-68E show a schematic of the sequences and oligonucleotidesused in the construction of the sucCD deletion.

FIG. 69 shows an exemplary BDO pathway. Enzyme names for each numberedstep are as follows: (1) alpha-ketoglutarate decarboxylase; (2)CoA-dependent succinate semialdehyde dehydrogenase; (3)4-hydroxybutyrate dehydrogenase; (4) 4-hydroxybutyryl-CoA transferase;(5) 4-hydroxybutyryl-CoA reductase; (6) 4-hydroxybutyraldehydereductase.

FIG. 70 shows an exemplary pathway for the formation of BDO fromglucose. Abbreviations: G6P-glucose-6-phosphate,PEP-phosphoenolpyruvate, PYR-pyruvate, OA-oxaloacetate,ACCOA-acetyl-CoA, CIT-citrate, ICIT-isocitrate, AKG-alpha-ketoglutarate,SUCCOA-succinyl-CoA, SUCSAL-succinate semialdehyde,4HB-4-hydroxybutyrate, 4HBCoA-4-hydroxybutyryl-CoA,4HBALD-4-hydroxybutyraldehyde, BDO-1,4-butanediol,GBL-gamma-butyrolactone. Genes of interest: ppc-PEP carboxylase,sucCD-succinyl-CoA synthetase, sucAB-lpdA-subunits of the AKGdehydrogenase complex, ybgC, tesB-acyl-CoenzymeA thioesterases

FIG. 71 shows the average BDO and 4HB numbers from four replicates of a4HB producing host strain that had the gene ppc overexpressed comparedwith the corresponding averages from four replicates of the controlstrain.

FIG. 72 shows the reduction in the average ethanol numbers from the fourreplicates of the strain that had ppc overexpressed and compared withthose from the control.

FIG. 73 shows the average BDO and 4HB numbers from four replicates of a4HB producing host strain that had the genes sucAB and mutant lpdAoverexpressed on pZS*compared with the corresponding averages from fourreplicates of the control strain. ALD and ADH were expressed on theplasmid pPZSX23R.

FIG. 74 shows the reduction in the average glutamate numbers from thefour replicates of the strain that had the genes sucAB and mutant lpdAoverexpressed on pZS* and compared with those from the control.

FIG. 75 shows apparent equilibrium constant for lactonization of 4-HB toGBL as a function of pH (at 22° C.) (from Efe et al., Biotechnol.Bioeng. 99:1392-1406 (2008)).

FIG. 76 shows the average BDO and 4HB numbers from four replicates of ahost strain capable of producing 4-hydrobutyryl-CoA that had theesterase integrated (2387) compared with the corresponding averages fromfour replicates of the control strain (2237).

FIG. 77 shows the average GBL numbers from four replicates of a hoststrain that had the esterase integrated (2387) compared with thecorresponding averages from four replicates of the control strain(2237). Lower GBL was observed with the strains that had the esteraseintegrated.

FIG. 78 shows the average BDO and 4HB numbers from four replicates of ahost strain that had the genes sucCD deleted (4269) compared with thecorresponding averages from four replicates of the control strain(4070).

FIG. 79 shows the average BDO and 4HB numbers from three replicates of ahost strain that had the genes ybgC and tesB deleted (1197) comparedwith the corresponding averages from three replicates of the controlstrain (1136).

FIG. 80 shows the average GBL numbers from three replicates of a hoststrain that had the genes ybgC and tesB deleted (1197) compared with thecorresponding averages from three replicates of the control strain(1136).

FIG. 81 shows the profile for the anion exchange elution, backfluxactivity, as well as the SDS page for the fractions.

FIG. 82 shows the profile from the size exclusion chromatogrpahy, thebackflux activity, as well as the SDS page for the fractions.

FIG. 83 shows backflux activity from the size exclusion column and theSDS page.

FIG. 84 shows the yjgB gene was cloned with a streptavidin tag,expressed and purified in order to characterize its properties. Theresults of the purification are via SDS-PAGE.

FIG. 85 shows yjgB activity in 100 mM BDO.

FIG. 86 shows the average BDO, 4HB and GBL numbers for the strain 1872versus strain 1889.

FIG. 87 shows the average BDO, 4HB and GBL numbers for the strain 956versus strain 879.

FIG. 88 shows the average BDO, 4HB and GBL numbers of the strains, 1889,2424, 2611 and 2471.

FIG. 89 shows the nucleotide sequence of the Yersinia gene (locusyinte0001_13710) (SEQ ID NO:177).

FIG. 90 shows the nucleotide sequence of the gene from Agrobacteriumtumefaciens (SEQ ID NO:178).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the design and production of cellsand organisms having biosynthetic production capabilities for4-hydroxybutanoic acid (4-HB), γ-butyrolactone, 1,4-butanediol (BDO),4-hydroxybutanal (4-HBal), 4-hydroxybutyryl-CoA (4-HBCoA) and/orputrescine. The invention, in particular, relates to the design ofmicrobial organisms capable of producing BDO, 4-HBal, 4-HBCoA and/orputrescine by introducing one or more nucleic acids encoding a BDO,4-HBal, 4-HBCoA and/or putrescine pathway enzyme.

In one embodiment, the invention utilizes in silico stoichiometricmodels of Escherichia coli metabolism that identify metabolic designsfor biosynthetic production of 4-hydroxybutanoic acid (4-HB),1,4-butanediol (BDO), 4-HBal, 4-HBCoA and/or putrescine. The resultsdescribed herein indicate that metabolic pathways can be designed andrecombinantly engineered to achieve the biosynthesis of 4-HBal, 4-HBCoAor 4-HB and downstream products such as 1,4-butanediol or putrescine inEscherichia coli and other cells or organisms. Biosynthetic productionof 4-HB, 4-HBal, 4-HBCoA, BDO and/or putrescine, for example, for the insilico designs can be confirmed by construction of strains having thedesigned metabolic genotype. These metabolically engineered cells ororganisms also can be subjected to adaptive evolution to further augment4-HB, 4-HBal, 4-HBCoA, BDO and/or putrescine biosynthesis, includingunder conditions approaching theoretical maximum growth.

In certain embodiments, the 4-HB, 4-HBal, 4-HBCoA, BDO and/or putrescinebiosynthesis characteristics of the designed strains make themgenetically stable and particularly useful in continuous bioprocesses.Separate strain design strategies were identified with incorporation ofdifferent non-native or heterologous reaction capabilities into E. colior other host organisms leading to 4-HB and 1,4-butanediol producingmetabolic pathways from either CoA-independent succinic semialdehydedehydrogenase, succinyl-CoA synthetase and CoA-dependent succinicsemialdehyde dehydrogenase, or glutamate:succinic semialdehydetransaminase. In silico metabolic designs were identified that resultedin the biosynthesis of 4-HB in both E. coli and yeast species from eachof these metabolic pathways. The 1,4-butanediol intermediateγ-butyrolactone can be generated in culture by spontaneous cyclizationunder conditions at pH<7.5, particularly under acidic conditions, suchas below pH 5.5, for example, pH<7, pH<6.5, pH<6, and particularly atpH<5.5 or lower.

Strains identified via the computational component of the platform canbe put into actual production by genetically engineering any of thepredicted metabolic alterations which lead to the biosyntheticproduction of 4-HB, 1,4-butanediol or other intermediate and/ordownstream products. In yet a further embodiment, strains exhibitingbiosynthetic production of these compounds can be further subjected toadaptive evolution to further augment product biosynthesis. The levelsof product biosynthesis yield following adaptive evolution also can bepredicted by the computational component of the system.

In other specific embodiments, microbial organisms were constructed toexpress a 4-HB biosynthetic pathway encoding the enzymatic steps fromsuccinate to 4-HB and to 4-HB-CoA. Co-expression of succinate coenzyme Atransferase, CoA-dependent succinic semialdehyde dehydrogenase,NAD-dependent 4-hydroxybutyrate dehydrogenase and 4-hydroxybutyratecoenzyme A transferase in a host microbial organism resulted insignificant production of 4-HB compared to host microbial organismslacking a 4-HB biosynthetic pathway. In a further specific embodiment,4-HB-producing microbial organisms were generated that utilizedα-ketoglutarate as a substrate by introducing nucleic acids encodingα-ketoglutarate decarboxylase and NAD-dependent 4-hydroxybutyratedehydrogenase.

In another specific embodiment, microbial organisms containing a1,4-butanediol (BDO) biosynthetic pathway were constructed thatbiosynthesized BDO when cultured in the presence of 4-HB. The BDObiosynthetic pathway consisted of a nucleic acid encoding either amultifunctional aldehyde/alcohol dehydrogenase or nucleic acids encodingan aldehyde dehydrogenase and an alcohol dehydrogenase. To supportgrowth on 4-HB substrates, these BDO-producing microbial organisms alsoexpressed 4-hydroxybutyrate CoA transferase or 4-butyrate kinase inconjunction with phosphotranshydroxybutyrlase. In yet a further specificembodiment, microbial organisms were generated that synthesized BDOthrough exogenous expression of nucleic acids encoding a functional 4-HBbiosynthetic pathway and a functional BDO biosynthetic pathway. The 4-HBbiosynthetic pathway consisted of succinate coenzyme A transferase,CoA-dependent succinic semialdehyde dehydrogenase, NAD-dependent4-hydroxybutyrate dehydrogenase and 4-hydroxybutyrate coenzyme Atransferase. The BDO pathway consisted of a multifunctionalaldehyde/alcohol dehydrogenase. Further described herein are additionalpathways for production of BDO (see FIGS. 8-13).

In a further embodiment, described herein is the cloning and expressionof a carboxylic acid reductase enzyme that functions in a4-hydroxybutanal, 4-hydroxybutyryl-CoA or 1,4-butanediol metabolicpathway. Advantages of employing a carboxylic acid reductase as opposedto an acyl-CoA reductase to form 4-hydroxybutyraldehyde(4-hydroxybutanal) include lower ethanol and GBL byproduct formationaccompanying the production of BDO. Also disclosed herein is theapplication of carboxylic acid reductase as part of additional numerouspathways to produce 1,4-butanediol and putrescine from the tricarboxylicacid (TCA) cycle metabolites, for example, succinate, succinyl-CoA,and/or alpha-ketoglutarate.

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism of the invention isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial organism's genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. Exemplarymetabolic polypeptides include enzymes or proteins within a biosyntheticpathway for a 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine family ofcompounds.

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, non-naturallyoccurring microorganisms can have genetic modifications to nucleic acidsencoding metabolic polypeptides or, functional fragments thereof.Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to amicrobial organism is intended to mean an organism that is substantiallyfree of at least one component as the referenced microbial organism isfound in nature. The term includes a microbial organism that is removedfrom some or all components as it is found in its natural environment.The term also includes a microbial organism that is removed from some orall components as the microbial organism is found in non-naturallyoccurring environments. Therefore, an isolated microbial organism ispartly or completely separated from other substances as it is found innature or as it is grown, stored or subsisted in non-naturally occurringenvironments. Specific examples of isolated microbial organisms includepartially pure microbes, substantially pure microbes and microbescultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or“microorganism” are intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

As used herein, the term “4-hydroxybutanoic acid” is intended to mean a4-hydroxy derivative of butyric acid having the chemical formula C₄H₈O₃and a molecular mass of 104.11 g/mol (126.09 g/mol for its sodium salt).The chemical compound 4-hydroxybutanoic acid also is known in the art as4-HB, 4-hydroxybutyrate, gamma-hydroxybutyric acid or GHB. The term asit is used herein is intended to include any of the compound's varioussalt forms and include, for example, 4-hydroxybutanoate and4-hydroxybutyrate. Specific examples of salt forms for 4-HB includesodium 4-HB and potassium 4-HB. Therefore, the terms 4-hydroxybutanoicacid, 4-HB, 4-hydroxybutyrate, 4-hydroxybutanoate, gamma-hydroxybutyricacid and GHB as well as other art recognized names are used synonymouslyherein.

As used herein, the term “monomeric” when used in reference to 4-HB isintended to mean 4-HB in a non-polymeric or underivatized form. Specificexamples of polymeric 4-HB include poly-4-hydroxybutanoic acid andcopolymers of, for example, 4-HB and 3-HB. A specific example of aderivatized form of 4-HB is 4-HB-CoA. Other polymeric 4-HB forms andother derivatized forms of 4-HB also are known in the art.

As used herein, the term “γ-butyrolactone” is intended to mean a lactonehaving the chemical formula C₄H₆O₂ and a molecular mass of 86.089 g/mol.The chemical compound γ-butyrolactone also is know in the art as GBL,butyrolactone, 1,4-lactone, 4-butyrolactone, 4-hydroxybutyric acidlactone, and gamma-hydroxybutyric acid lactone. The term as it is usedherein is intended to include any of the compound's various salt forms.

As used herein, the term “1,4-butanediol” is intended to mean an alcoholderivative of the alkane butane, carrying two hydroxyl groups which hasthe chemical formula C₄H₁₀O₂ and a molecular mass of 90.12 g/mol. Thechemical compound 1,4-butanediol also is known in the art as BDO and isa chemical intermediate or precursor for a family of compounds referredto herein as BDO family of compounds.

As used herein, the term “4-hydroxybutanal” is intended to mean analedehyde having the chemical formula C₄H₈O₂ and a molecular mass of88.10512 g/mol. The chemical compound 4-hydroxybutanal (4-HBal) is alsoknown in the art as 4-hydroxybutyraldehyde.

As used herein, the term “putrescine” is intended to mean a diaminehaving the chemical formula C₄H₁₂N₂ and a molecular mass of 88.15148g/mol. The chemical compound putrescine is also known in the art as1,4-butanediamine, 1,4-diaminobutane, butylenediamine,tetramethylenediamine, tetramethyldiamine, and 1,4-butylenediamine.

As used herein, the term “tetrahydrofuran” is intended to mean aheterocyclic organic compound corresponding to the fully hydrogenatedanalog of the aromatic compound furan which has the chemical formulaC₄H₈O and a molecular mass of 72.11 g/mol. The chemical compoundtetrahydrofuran also is known in the art as THF, tetrahydrofuran,1,4-epoxybutane, butylene oxide, cyclotetramethylene oxide,oxacyclopentane, diethylene oxide, oxolane, furanidine, hydrofuran,tetra-methylene oxide. The term as it is used herein is intended toinclude any of the compound's various salt forms.

As used herein, the term “CoA” or “coenzyme A” is intended to mean anorganic cofactor or prosthetic group (nonprotein portion of an enzyme)whose presence is required for the activity of many enzymes (theapoenzyme) to form an active enzyme system. Coenzyme A functions incertain condensing enzymes, acts in acetyl or other acyl group transferand in fatty acid synthesis and oxidation, pyruvate oxidation and inother acetylation.

As used herein, the term “substantially anaerobic” when used inreference to a culture or growth condition is intended to mean that theamount of oxygen is less than about 10% of saturation for dissolvedoxygen in liquid media. The term also is intended to include sealedchambers of liquid or solid medium maintained with an atmosphere of lessthan about 1% oxygen.

The non-naturally occurring microbal organisms of the invention cancontain stable genetic alterations, which refers to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications will be greater than 50generations, including indefinitely.

In the case of gene disruptions, a particularly useful stable geneticalteration is a gene deletion. The use of a gene deletion to introduce astable genetic alteration is particularly useful to reduce thelikelihood of a reversion to a phenotype prior to the geneticalteration. For example, if desired, stable growth-coupled production ofa biochemical can be achieved, for example, by deletion of a geneencoding an enzyme catalyzing one or more reactions within a set ofmetabolic modifications. The stability of growth-coupled production of abiochemical can be further enhanced through multiple deletions,significantly reducing the likelihood of multiple compensatoryreversions occurring for each disrupted activity.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein are described withreference to a suitable source or host organism such as E. coli, yeast,or other organisms disclosed herein and their corresponding metabolicreactions or a suitable source organism for desired genetic materialsuch as genes encoding enzymes for their corresponding metabolicreactions for a desired metabolic pathway. However, given the completegenome sequencing of a wide variety of organisms and the high level ofskill in the area of genomics, those skilled in the art will readily beable to apply the teachings and guidance provided herein to essentiallyall other organisms. For example, the E. coli metabolic alterationsexemplified herein can readily be applied to other species byincorporating the same or analogous encoding nucleic acid from speciesother than the referenced species. Such genetic alterations include, forexample, genetic alterations of species homologs, in general, and inparticular, orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less that 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities. Members of theserine protease family of enzymes, including tissue plasminogenactivator and elastase, are considered to have arisen by verticaldescent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the production, includinggrowth-coupled production, of a biochemical product, those skilled inthe art will understand that the orthologous gene harboring themetabolic activity to be introduced or disrupted is to be chosen forconstruction of the non-naturally occurring microorganism. An example oforthologs exhibiting separable activities is where distinct activitieshave been separated into distinct gene products between two or morespecies or within a single species. A specific example is the separationof elastase proteolysis and plasminogen proteolysis, two types of serineprotease activity, into distinct molecules as plasminogen activator andelastase. A second example is the separation of mycoplasma 5′-3′exonuclease and Drosophila DNA polymerase III activity. The DNApolymerase from the first species can be considered an ortholog toeither or both of the exonuclease or the polymerase from the secondspecies and vice versa.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor. Groups of paralogousprotein families include HipA homologs, luciferase genes, peptidases,and others.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene productcompared to a gene encoding the function sought to be substituted.Therefore, a nonorthologous gene includes, for example, a paralog or anunrelated gene.

Therefore, in identifying and constructing the non-naturally occurringmicrobial organisms of the invention having 4-HB, GBL, 4-HBal, 4-HBCoA,BDO and/or putrescine biosynthetic capability, those skilled in the artwill understand with applying the teaching and guidance provided hereinto a particular species that the identification of metabolicmodifications can include identification and inclusion or inactivationof orthologs. To the extent that paralogs and/or nonorthologous genedisplacements are present in the referenced microorganism that encode anenzyme catalyzing a similar or substantially similar metabolic reaction,those skilled in the art also can utilize these evolutionally relatedgenes. Similarly for a gene disruption, evolutionally related genes canalso be disrupted or deleted in a host microbial organism to reduce oreliminate functional redundancy of enzymatic activities targeted fordisruption.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50;expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

Disclosed herein are non-naturally occurring microbial biocatalyst ormicrobial organisms including a microbial organism having a4-hydroxybutanoic acid (4-HB) biosynthetic pathway that includes atleast one exogenous nucleic acid encoding 4-hydroxybutanoatedehydrogenase, CoA-independent succinic semialdehyde dehydrogenase,succinyl-CoA synthetase, CoA-dependent succinic semialdehydedehydrogenase, glutamate: succinic semialdehyde transaminase,alpha-ketoglutarate decarboxylase, or glutamate decarboxylase, whereinthe exogenous nucleic acid is expressed in sufficient amounts to producemonomeric 4-hydroxybutanoic acid (4-HB). 4-hydroxybutanoatedehydrogenase is also referred to as 4-hydroxybutyrate dehydrogenase or4-HB dehydrogenase. Succinyl-CoA synthetase is also referred to assuccinyl-CoA synthase or succinyl-CoA ligase.

Also disclosed herein is a non-naturally occurring microbial biocatalystor microbial organism including a microbial organism having a4-hydroxybutanoic acid (4-HB) biosynthetic pathway having at least oneexogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase,succinyl-CoA synthetase, CoA-dependent succinic semialdehydedehydrogenase, or α-ketoglutarate decarboxylase, wherein the exogenousnucleic acid is expressed in sufficient amounts to produce monomeric4-hydroxybutanoic acid (4-HB).

The non-naturally occurring microbial biocatalysts or microbialorganisms can include microbial organisms that employ combinations ofmetabolic reactions for biosynthetically producing the compounds of theinvention. The biosynthesized compounds can be produced intracellularlyand/or secreted into the culture medium. Exemplary compounds produced bythe non-naturally occurring microorganisms include, for example,4-hydroxybutanoic acid, 1,4-butanediol and γ-butyrolactone.

In one embodiment, a non-naturally occurring microbial organism isengineered to produce 4-HB. This compound is one useful entry point intothe 1,4-butanediol family of compounds. The biochemical reactions forformation of 4-HB from succinate, from succinate through succinyl-CoA orfrom α-ketoglutarate are shown in steps 1-8 of FIG. 1.

It is understood that any combination of appropriate enzymes of a BDO,4-HBal, 4-HBCoA and/or putrescine pathway can be used so long asconversion from a starting component to the BDO, 4-HBal, 4-HBCoA and/orputrescine product is achieved. Thus, it is understood that any of themetabolic pathways disclosed herein can be utilized and that it is wellunderstood to those skilled in the art how to select appropriate enzymesto achieve a desired pathway, as disclosed herein.

In another embodiment, disclosed herein is a non-naturally occurringmicrobial organism, comprising a microbial organism having a1,4-butanediol (BDO) pathway comprising at least one exogenous nucleicacid encoding a BDO pathway enzyme expressed in a sufficient amount toproduce BDO, the BDO pathway comprising 4-aminobutyrate CoA transferase,4-aminobutyryl-CoA hydrolase, 4-aminobutyrate-CoA ligase,4-aminobutyryl-CoA oxidoreductase (deaminating), 4-aminobutyryl-CoAtransaminase, or 4-hydroxybutyryl-CoA dehydrogenase (see Example VIITable 17). The BDO pathway further can comprise 4-hydroxybutyryl-CoAreductase (alcohol forming), 4-hydroxybutyryl-CoA reductase, or1,4-butanediol dehydrogenase.

It is understood by those skilled in the art that various combinationsof the pathways can be utilized, as disclosed herein. For example, in anon-naturally occurring microbial organism, the nucleic acids can encode4-aminobutyrate CoA transferase, 4-aminobutyryl-CoA hydrolase, or4-aminobutyrate-CoA ligase; 4-aminobutyryl-CoA oxidoreductase(deaminating) or 4-aminobutyryl-CoA transaminase; and4-hydroxybutyryl-CoA dehydrogenase. Other exemplary combinations arespecifically describe below and further can be found in FIGS. 8-13. Forexample, the BDO pathway can further comprise 4-hydroxybutyryl-CoAreductase (alcohol forming), 4-hydroxybutyryl-CoA reductase, or1,4-butanediol dehydrogenase.

Additionally disclosed herein is a non-naturally occurring microbialorganism, comprising a microbial organism having a BDO pathwaycomprising at least one exogenous nucleic acid encoding a BDO pathwayenzyme expressed in a sufficient amount to produce BDO, the BDO pathwaycomprising 4-aminobutyrate CoA transferase, 4-aminobutyryl-CoAhydrolase, 4-aminobutyrate-CoA ligase, 4-aminobutyryl-CoA reductase(alcohol forming), 4-aminobutyryl-CoA reductase, 4-aminobutan-1-oldehydrogenase, 4-aminobutan-1-ol oxidoreductase (deaminating) or4-aminobutan-1-ol transaminase (see Example VII and Table 18), and canfurther comprise 1,4-butanediol dehydrogenase. For example, theexogenous nucleic acids can encode 4-aminobutyrate CoA transferase,4-aminobutyryl-CoA hydrolase, or 4-aminobutyrate-CoA ligase;4-aminobutyryl-CoA reductase (alcohol forming); and 4-aminobutan-1-oloxidoreductase (deaminating) or 4-aminobutan-1-ol transaminase. Inaddition, the nucleic acids can encode. 4-aminobutyrate CoA transferase,4-aminobutyryl-CoA hydrolase, or 4-aminobutyrate-CoA ligase;4-aminobutyryl-CoA reductase; 4-aminobutan-1-ol dehydrogenase; and4-aminobutan-1-ol oxidoreductase (deaminating) or 4-aminobutan-1-oltransaminase.

Also disclosed herein is a non-naturally occurring microbial organism,comprising a microbial organism having a BDO pathway comprising at leastone exogenous nucleic acid encoding a BDO pathway enzyme expressed in asufficient amount to produce BDO, the BDO pathway comprising4-aminobutyrate kinase, 4-aminobutyraldehyde dehydrogenase(phosphorylating), 4-aminobutan-1-ol dehydrogenase,4-aminobutan-1-oloxidoreductase (deaminating), 4-aminobutan-1-oltransaminase, [(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase(deaminating), [(4-aminobutanolyl)oxy]phosphonic acid transaminase,4-hydroxybutyryl-phosphate dehydrogenase, or 4-hydroxybutyraldehydedehydrogenase (phosphorylating) (see Example VII and Table 19). Forexample, the exogenous nucleic acids can encode 4-aminobutyrate kinase;4-aminobutyraldehyde dehydrogenase (phosphorylating); 4-aminobutan-1-oldehydrogenase; and 4-aminobutan-1-ol oxidoreductase (deaminating) or4-aminobutan-1-ol transaminase. Alternatively, the exogenous nucleicacids can encode 4-aminobutyrate kinase;[(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase (deaminating) or[(4-aminobutanolyl)oxy]phosphonic acid transaminase;4-hydroxybutyryl-phosphate dehydrogenase; and 4-hydroxybutyraldehydedehydrogenase (phosphorylating).

Additionally disclosed herein is a non-naturally occurring microbialorganism, comprising a microbial organism having a BDO pathwaycomprising at least one exogenous nucleic acid encoding a BDO pathwayenzyme expressed in a sufficient amount to produce BDO, the BDO pathwaycomprising alpha-ketoglutarate 5-kinase, 2,5-dioxopentanoic semialdehydedehydrogenase (phosphorylating), 2,5-dioxopentanoic acid reductase,alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase,alpha-ketoglutaryl-CoA ligase, alpha-ketoglutaryl-CoA reductase,5-hydroxy-2-oxopentanoic acid dehydrogenase, alpha-ketoglutaryl-CoAreductase (alcohol forming), 5-hydroxy-2-oxopentanoic aciddecarboxylase, or 5-hydroxy-2-oxopentanoic acid dehydrogenase(decarboxylation) (see Example VIII and Table 20). The BDO pathway canfurther comprise 4-hydroxybutyryl-CoA reductase (alcohol forming),4-hydroxybutyryl-CoA reductase, or 1,4-butanediol dehydrogenase. Forexample, the exogenous nucleic acids can encode alpha-ketoglutarate5-kinase; 2,5-dioxopentanoic semialdehyde dehydrogenase(phosphorylating); 2,5-dioxopentanoic acid reductase; and5-hydroxy-2-oxopentanoic acid decarboxylase. Alternatively, theexogenous nucleic acids can encode alpha-ketoglutarate 5-kinase;2,5-dioxopentanoic semialdehyde dehydrogenase (phosphorylating);2,5-dioxopentanoic acid reductase; and 5-hydroxy-2-oxopentanoic aciddehydrogenase (decarboxylation). Alternatively, the exogenous nucleicacids can encode alpha-ketoglutarate CoA transferase,alpha-ketoglutaryl-CoA hydrolase, or alpha-ketoglutaryl-CoA ligase;alpha-ketoglutaryl-CoA reductase, 5-hydroxy-2-oxopentanoic aciddehydrogenase; and 5-hydroxy-2-oxopentanoic acid decarboxylase. Inanother embodiment, the exogenous nucleic acids can encodealpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase,or alpha-ketoglutaryl-CoA ligase; alpha-ketoglutaryl-CoA reductase,5-hydroxy-2-oxopentanoic acid dehydrogenase, and5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation).Alternatively, the exogenous nucleic acids can encodealpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase,or alpha-ketoglutaryl-CoA ligase; alpha-ketoglutaryl-CoA reductase(alcohol forming); and 5-hydroxy-2-oxopentanoic acid decarboxylase. Inyet another embodiment, the exogenous nucleic acids can encodealpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase,or alpha-ketoglutaryl-CoA ligase; alpha-ketoglutaryl-CoA reductase(alcohol forming); and 5-hydroxy-2-oxopentanoic acid dehydrogenase(decarboxylation).

Further disclosed herein is a non-naturally occurring microbialorganism, comprising a microbial organism having a BDO pathwaycomprising at least one exogenous nucleic acid encoding a BDO pathwayenzyme expressed in a sufficient amount to produce BDO, the BDO pathwaycomprising glutamate CoA transferase, glutamyl-CoA hydrolase,glutamyl-CoA ligase, glutamate 5-kinase, glutamate-5-semialdehydedehydrogenase (phosphorylating), glutamyl-CoA reductase,glutamate-5-semialdehyde reductase, glutamyl-CoA reductase (alcoholforming), 2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating),2-amino-5-hydroxypentanoic acid transaminase, 5-hydroxy-2-oxopentanoicacid decarboxylase, 5-hydroxy-2-oxopentanoic acid dehydrogenase(decarboxylation) (see Example IX and Table 21). For example, theexogenous nucleic acids can encode glutamate CoA transferase,glutamyl-CoA hydrolase, or glutamyl-CoA ligase; glutamyl-CoA reductase;glutamate-5-semialdehyde reductase; 2-amino-5-hydroxypentanoic acidoxidoreductase (deaminating) or 2-amino-5-hydroxypentanoic acidtransaminase; and 5-hydroxy-2-oxopentanoic acid decarboxylase or5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation).Alternatively, the exogenous nucleic acids can encode glutamate5-kinase; glutamate-5-semialdehyde dehydrogenase (phosphorylating);glutamate-5-semialdehyde reductase; 2-amino-5-hydroxypentanoic acidoxidoreductase (deaminating) or 2-amino-5-hydroxypentanoic acidtransaminase; and 5-hydroxy-2-oxopentanoic acid decarboxylase or5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation). In stillanother embodiment, the exogenous nucleic acids can encode glutamate CoAtransferase, glutamyl-CoA hydrolase, or glutamyl-CoA ligase;glutamyl-CoA reductase (alcohol forming); 2-amino-5-hydroxypentanoicacid oxidoreductase (deaminating) or 2-amino-5-hydroxypentanoic acidtransaminase; and 5-hydroxy-2-oxopentanoic acid decarboxylase or5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation). In yetanother embodiment, the exogenous nucleic acids can encode glutamate5-kinase; glutamate-5-semialdehyde dehydrogenase (phosphorylating);2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or2-amino-5-hydroxypentanoic acid transaminase; and5-hydroxy-2-oxopentanoic acid decarboxylase or 5-hydroxy-2-oxopentanoicacid dehydrogenase (decarboxylation).

Also disclosed herein is a non-naturally occurring microbial organism,comprising a microbial organism having a BDO pathway comprising at leastone exogenous nucleic acid encoding a BDO pathway enzyme expressed in asufficient amount to produce BDO, the BDO pathway comprising3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase,vinylacetyl-CoA Δ-isomerase, or 4-hydroxybutyryl-CoA dehydratase (seeExample X and Table 22). For example, the exogenous nucleic acids canencode 3-hydroxybutyryl-CoA dehydrogenase; 3-hydroxybutyryl-CoAdehydratase; vinylacetyl-CoA Δ-isomerase; and 4-hydroxybutyryl-CoAdehydratase.

Further disclosed herein is a non-naturally occurring microbialorganism, comprising a microbial organism having a BDO pathwaycomprising at least one exogenous nucleic acid encoding a BDO pathwayenzyme expressed in a sufficient amount to produce BDO, the BDO pathwaycomprising homoserine deaminase, homoserine CoA transferase,homoserine-CoA hydrolase, homoserine-CoA ligase, homoserine-CoAdeaminase, 4-hydroxybut-2-enoyl-CoA transferase,4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA ligase,4-hydroxybut-2-enoate reductase, 4-hydroxybutyryl-CoA transferase,4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or4-hydroxybut-2-enoyl-CoA reductase (see Example XI and Table 23). Forexample, the exogenous nucleic acids can encode homoserine deaminase;4-hydroxybut-2-enoyl-CoA transferase, 4-hydroxybut-2-enoyl-CoAhydrolase, 4-hydroxybut-2-enoyl-CoA ligase; 4-hydroxybut-2-enoyl-CoAreductase. Alternatively, the exogenous nucleic acids can encodehomoserine CoA transferase, homoserine-CoA hydrolase, or homoserine-CoAligase; homoserine-CoA deaminase; and 4-hydroxybut-2-enoyl-CoAreductase. In a further embodiment, the exogenous nucleic acids canencode homoserine deaminase; 4-hydroxybut-2-enoate reductase; and4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA hydrolase, or4-hydroxybutyryl-CoA ligase. Alternatively, the exogenous nucleic acidscan encode homoserine CoA transferase, homoserine-CoA hydrolase, orhomoserine-CoA ligase; homoserine-CoA deaminase; and4-hydroxybut-2-enoyl-CoA reductase.

Further disclosed herein is a non-naturally occurring microbialorganism, comprising a microbial organism having a BDO pathwaycomprising at least one exogenous nucleic acid encoding a BDO pathwayenzyme expressed in a sufficient amount to produce BOD, the BDO pathwaycomprising succinyl-CoA reductase (alcohol forming),4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase,4-hydroxybutanal dehydrogenase (phosphorylating) (see Table 15). Such aBDO pathway can further comprise succinyl-CoA reductase,4-hydroxybutyrate dehydrogenase, 4-hydroxybutyryl-CoA transferase,4-hydroxybutyrate kinase, phosphotrans-4-hydroxybutyrylase,4-hydroxybutyryl-CoA reductase, 4-hydroxybutyryl-CoA reductase (alcoholforming), or 1,4-butanediol dehydrogenase.

Additionally disclosed herein is a non-naturally occurring microbialorganism, comprising a microbial organism having a BDO pathwaycomprising at least one exogenous nucleic acid encoding a BDO pathwayenzyme expressed in a sufficient amount to produce BDO, the BDO pathwaycomprising glutamate dehydrogenase, 4-aminobutyrate oxidoreductase(deaminating), 4-aminobutyrate transaminase, glutamate decarboxylase,4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase,4-hydroxybutanal dehydrogenase (phosphorylating) (see Table 16). Such aBDO pathway can further comprise alpha-ketoglutarate decarboxylase,4-hydroxybutyrate dehydrogenase, 4-hydroxybutyryl-CoA transferase,4-hydroxybutyrate kinase, phosphotrans-4-hydroxybutyrylase,4-hydroxybutyryl-CoA reductase, 4-hydroxybutyryl-CoA reductase (alcoholforming), or 1,4-butanediol dehydrogenase.

The pathways described above are merely exemplary. One skilled in theart can readily select appropriate pathways from those disclosed hereinto obtain a suitable BDO pathway or other metabolic pathway, as desired.

The invention provides genetically modified organisms that allowimproved production of a desired product such as BDO by increasing theproduct or decreasing undesirable byproducts. As disclosed herein, theinvention provides a non-naturally occurring microbial organism,comprising a microbial organism having a 1,4-butanediol (BDO) pathwaycomprising at least one exogenous nucleic acid encoding a BDO pathwayenzyme expressed in a sufficient amount to produce BDO. In oneembodiment, the microbial organism is genetically modified to expressexogenous succinyl-CoA synthetase (see Example XII). For example, thesuccinyl-CoA synthetase can be encoded by an Escherichia coli sucCDgenes.

In another embodiment, the microbial organism is genetically modified toexpress exogenous alpha-ketoglutarate decarboxylase (see Example XIII).For example, the alpha-ketoglutarate decarboxylase can be encoded by theMycobacterium bovis sucA gene. In still another embodiment, themicrobial organism is genetically modified to express exogenoussuccinate semialdehyde dehydrogenase and 4-hydroxybutyrate dehydrogenaseand optionally 4-hydroxybutyryl-CoA/acetyl-CoA transferase (see ExampleXIII). For example, the succinate semialdehyde dehydrogenase(CoA-dependent), 4-hydroxybutyrate dehydrogenase and4-hydroxybutyryl-CoA/acetyl-CoA transferase can be encoded byPorphyromonas gingivalis W83 genes. In an additional embodiment, themicrobial organism is genetically modified to express exogenous butyratekinase and phosphotransbutyrylase (see Example XIII). For example, thebutyrate kinase and phosphotransbutyrylase can be encoded by Clostridiumacetobutilicum buk1 and ptb genes.

In yet another embodiment, the microbial organism is geneticallymodified to express exogenous 4-hydroxybutyryl-CoA reductase (seeExample XIII). For example, the 4-hydroxybutyryl-CoA reductase can beencoded by Clostridium beijerinckii ald gene. Additionally, in anembodiment of the invention, the microbial organism is geneticallymodified to express exogenous 4-hydroxybutanal reductase (see ExampleXIII). For example, the 4-hydroxybutanal reductase can be encoded byGeobacillus thermoglucosidasius adh1 gene. In another embodiment, themicrobial organism is genetically modified to express exogenous pyruvatedehydrogenase subunits (see Example XIV). For example, the exogenouspyruvate dehydrogenase can be NADH insensitive. The pyruvatedehydrogenase subunit can be encoded by the Klebsiella pneumonia lpdAgene. In a particular embodiment, the pyruvate dehydrogenase subunitgenes of the microbial organism can be under the control of a pyruvateformate lyase promoter.

In still another embodiment, the microbial organism is geneticallymodified to disrupt a gene encoding an aerobic respiratory controlregulatory system (see Example XV). For example, the disruption can beof the arcA gene. Such an organism can further comprise disruption of agene encoding malate dehydrogenase. In a further embodiment, themicrobial organism is genetically modified to express an exogenous NADHinsensitive citrate synthase (see Example XV). For example, the NADHinsensitive citrate synthase can be encoded by gltA, such as an R163Lmutant of gltA. In still another embodiment, the microbial organism isgenetically modified to express exogenous phosphoenolpyruvatecarboxykinase (see Example XVI). For example, the phosphoenolpyruvatecarboxykinase can be encoded by an Haemophilus influenzaphosphoenolpyruvate carboxykinase gene.

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism having a 4-hydroxybutyrate pathway andbeing capable of producing 4-hydroxybutyrate, wherein the microbialorganism comprises a genetic modification that increases expression ofphosphoenolpyruvate carboxylase (see Example XXXI). In such anembodiment in which phosphoenolpyruvate expression is increased, themicrobial organism can exhibit decreased production of ethanol, acetate,pyruvate or alanine, or a combination thereof, relative to a parentmicrobial organism in the absence of the genetic modification. Asdisclosed herein, the overexpression of posphoenolpyruvate (PEP)carboxylase leads to conversion of more phosphoenolpyruvate intooxaloacetate, thereby reducing flux from PEP into pyruvate andsubsequently into acetyl-CoA (Example XXXI). Reducing flux from PEP topyruvate and acetyl-CoA increases flux into the TCA cycle andconsequently a 4HB or BDO pathway.

In yet another embodiment, the invention provides a non-naturallyoccurring microbial organism having a 4-hydroxybutyrate pathway andbeing capable of producing 4-hydroxybutyrate, wherein the microbialorganism comprises a genetic modification that increases expression ofalpha-ketoglutarate dehydrogenase (see Example XXXII). The increasedexpression of alpha-ketoglutarate dehydrogenase can result in decreasedproduction of glutamate relative to a parent microbial organism in theabsence of the genetic modification. Such a microbial organism can alsoexhibit decreased production of ethanol, acetate, pyruvate or alanine,or a combination thereof, relative to a parent microbial organism in theabsence of the genetic modification. As disclosed herein, the formationof glutamate can lead to a carbon loss and reduction in yield.Therefore, increased expression of alpha-ketoglutarate dehydrogenase canreduce glutamate as well as other by-products such as ethanol, acetate,alanine and/or pyruvate.

In a further embodiment, the invention provides a non-naturallyoccurring microbial organism having a 4-hydroxybutyrate pathway andbeing capable of producing 4-hydroxybutyrate, wherein the microbialorganism comprises a genetic modification that increases expression of anon-phosphotransferase (PTS) glucose uptake system (see Example XXXIII).In such an embodiment, the genetic modification can comprise increasedexpression of a permease, glucokinase, or a glucose facilitator, or acombination thereof. In addition, such a microbial organism can exhibitdecreased production of ethanol, acetate, pyruvate, or alanine, or acombination thereof, relative to a parent microbial organism in theabsence of the genetic modification. The introduction of a non-PTSsucrose uptake system has been previously described in U.S. publication2011/0045575 as a way to reduce pyruvate formation in a microbialorganism utilizing sucrose as a carbon source. In contrast, theutilization of a non-PTS glucose uptake system, as described herein, isto provide a better balance between the available oxaloacetate incomparison to acetyl-CoA (see Example XXXIII).

In another embodiment, the invention provides a non-naturally occurringmicrobial organism having a 4-hydroxybutyrate pathway and being capableof producing 4-hydroxybutyrate, wherein the microbial organism comprisesa genetic modification that increases expression of agamma-butyrolactone esterase (Example XXXIV). Such a microbial organismcan exhibit decreased production of gamma-butyrolactone relative to aparent microbial organism in the absence of the genetic modification. Asdisclosed herein, gamma-butyrolactone is a byproduct formed during thefermentation of sugars to 1,4-butanediol. Gamma-butyrolactone can formfrom the unstable pathway intermediate 4-hydroxybutyryl-CoA as well asspontaneous lactonization of 4-hydroxybutyrate. As disclosed herein, theexpression of a gamma-butyrolactone can accelerate the hydrolysis ofgamma-butyrolactone to BDO, thereby improving BDO product yield andeliminating a byproduct (see Example XXXIV).

In additional embodiments disclosed herein, a genetic modification caninclude gene disruptions or deletions to decrease expression of anenzyme. For example, in a further embodiment, the invention provides anon-naturally occurring microbial organism having a 4-hydroxybutyratepathway and being capable of producing 4-hydroxybutyrate, wherein themicrobial organism comprises a genetic modification that decreasesexpression of succinyl-CoA synthetase (Example XXXV). In such anembodiment, the microbial organism can exhibit increased production of4-hydroxybutyrate relative to a parent microbial organism in the absenceof the genetic modification. As described herein, repeated rounds offlux through the TCA cycle results in carbon loss as CO₂. The deletionof succinyl-CoA synthetase blocks the TCA cycle downstream ofsuccinyl-CoA, thereby reducing CO₂ loss (see Example XXXV).

In a further embodiment, the invention provides a non-naturallyoccurring microbial organism having a 4-hydroxybutyrate pathway andbeing capable of producing 4-hydroxybutyrate, wherein the microbialorganism comprises a genetic modification that decreases expression ofan acyl coenzyme A thioesterase (see Example XXXVI). In such anembodiment the microbial organism can exhibit decreased production ofgamma-butyrolactone relative to a parent microbial organism in theabsence of the genetic modification. In yet a further embodiment, such amicrobial organism can comprise at least two genetic modifications thatdecrease expression of at least two acyl coenzyme A thioesterases. Asdisclosed herein, the BDO pathway intermediate 4-hydroxybutyryl-CoAspontaneously and enzymatically cyclizes to form gamma-butyrolactone(GBL). By deleting acyl coenzyme-A thioesterases, the formation of thebyproduct GBL was reduced (see Example XXXVI).

In another embodiment, the invention provides a non-naturally occurringmicrobial organism having a 4-hydroxybutyrate pathway and being capableof producing 4-hydroxybutyrate, wherein the microbial organism comprisesa genetic modification that decreases expression of an alcoholdehydrogenase (see Example XXVII). In such an embodiment, the microbialorganism can exhibit decreased backflux from a downstream product of the4-hydroxybutyrate pathway relative to a parent microbial organism in theabsence of said genetic modification. At high titers of BDO, adownstream product from 4-hydroxybutyrate, the high concentrations ofproduct can result in backflux within the pathway or through sidereactions, thereby resulting in a decreased product yield. As describedherein, several endogenous alcohol dehydrogenases were found tocontribute to backflux, whereas deletion of one or more of theendogenous alcohol dehydrogenases decreased backflux without reducingproduction of 4-hydroxybutyrate or BDO, and in fact increased BDOformation (see Example XXVII).

In still another embodiment, the invention provides a non-naturallyoccurring microbial organism having a 4-hydroxybutyrate pathway andbeing capable of producing 4-hydroxybutyrate, wherein the microbialorganism comprises a genetic modification that decreases expression of anon-energy-producing NADH dehydrogenase (see Example XXXVIII). Thedecreased expression of a non-energy-producging NADH dehydrogenasesuppresses depletion of the NADH pool resulting from the activity of thenon-energy-producing NADH dehydrogenase. Additionally, the microbialorganisms exhibit increased energy efficiency in the microbial organismrelative to a parent microbial organism in the absence of the geneticmodification. As described herein, the electron transport chain hasmultiple NADH dehydrogenases and cytochrome oxidases with varyingability to translocate protons. Some NADH dehydrogenases consume NADHwithout linking the consumption to proton translocation and energyproduction, and such NADH dehydrogenases are considered to benon-energy-producing NADH dehydrogenases. One exemplarynon-energy-producing NADH dehydrogenase is NADH II of E. coli (seeExample XXXVIII). Additional genes encoding enzymes with non-energyproducing NAD(P)H dehydrogenase activities include wrbA, yieF, and kefF.One skilled in the art will readily understand the meaning of anon-energy-producing NADH dehydrogenase as one that does not couple NADHoxidation to electron transport and proton translocation and theformation of ATP. By decreasing expression of a non-energy-producingNADH dehydrogenase, the depletion of the NADH pool within the cell issuppressed, thereby making the cell more energy efficient and/orallowing NADH to be utilized in desired synthetic pathways.

In a further embodiment, the invention provides a non-naturallyoccurring microbial organism having a 4-hydroxybutyrate pathway andbeing capable of producing 4-hydroxybutyrate, wherein the microbialorganism comprises a genetic modification that decreases expression of acytochrome oxidase (see Example XXXIX). In such an embodiment, themicrobial organism can exhibit increased energy efficiency relative to aparent microbial organism in the absence of the genetic modification. Asdisclosed herein, cytochrome oxidases involved in the electron transportchain have different energy-conserving efficiencies. By decreasingexpression of one or more cytochrome oxidases, the energy efficiency ofthe cell can be increased (see Example XXXIX). Even in a case where anincrease in product yield is not observed under certain conditions (seeExample XXXIX), such genetic modifications can be advantageous inproviding a greater tolerance for a range of oxygen concentrations, inparticular producing a product more efficiently in a large fermentor,where the available oxygen varies within the ferementor. By improvingthe energy efficiency of the microbial organism, the organism cantolerate lower oxygen conditions since need for energy production fromthe electron transport chain is reduced. Thus, in a particularembodiment of the invention, the microbial organism can exhibit anincreased tolerance to a range of oxygen concentrations relative to aparent microbial organism in the absence of said genetic modification.

The invention thus provides a non-naturally occurring microbial organismhaving a 4-hydroxybutyrate pathway and being capable of producing4-hydroxybutyrate, wherein the microbial organism comprises a geneticmodification, the genetic modification selected from: (A) a geneticmodification that increases expression of phosphoenolpyruvatecarboxylase; (B) a genetic modification that increases expression ofalpha-ketoglutarate dehydrogenase; (C) a genetic modification thatincreases expression of a non-phosphotransferase (PTS) glucose uptakesystem; (D) a genetic modification that increases expression of agamma-butyrolactone esterase; (E) a genetic modification that decreasesexpression of succinyl-CoA synthetase; (F) a genetic modification thatdecreases expression of an acyl coenzyme A thioesterase; (G) a geneticmodification that decreases expression of an alcohol dehydrogenase; (H)a genetic modification that decreases expression of anon-energy-producing NADH dehydrogenase; (I) a genetic modification thatdecreases expression of a cytochrome oxidase; and (J) a combination oftwo or more of the genetic modifications of parts (A)-(I). In furtherparticular embodiments, (K) the microbial organism of part (A), (B), or(C) has decreased production of ethanol, acetate, pyruvate or alanine,or a combination thereof, relative to a parent microbial organism in theabsence of said genetic modification; (L) the microbial organism of part(B) has decreased production of glutamate relative to a parent microbialorganism in the absence of said genetic modification; (M) the microbialorganism of part (C) has a genetic modification comprising increasedexpression of a permease, glucokinase, or a glucose facilitator, or acombination thereof; (N) the microbial organism of part (D) hasdecreased production of gamma-butyrolactone relative to a parentmicrobial organism in the absence of said genetic modification; (O) themicrobial organism of part (E) has increased production of4-hydroxybutyrate relative to a parent microbial organism in the absenceof said genetic modification; (P) the microbial organism of part (F) hasdecreased production of gamma-butyrolactone relative to a parentmicrobial organism in the absence of said genetic modification; (Q) themicrobial organism of part (F) has a genetic modification comprising atleast two genetic modifications that decrease expression of at least twoacyl coenzyme A thioesterases; (R) the microbial organism of part (G)has decreased backflux from a downstream product of the4-hydroxybutyrate pathway relative to a parent microbial organism in theabsence of said genetic modification; (S) the microbial organism of part(H) has suppressed depletion of the NADH pool or increased energyefficiency in the microbial organism, or a combination thereof, relativeto a parent microbial organism in the absence of said geneticmodification; (T) the microbial organism of part (I) has increasedenergy efficiency relative to a parent microbial organism in the absenceof said genetic modification; or (U) the microbial organism of part (I)has increased tolerance to a range of oxygen concentrations relative toa parent microbial organism in the absence of said genetic modification.In a further embodiment, the invention provides a microbial organism,where the microbial organism of parts (D) or (F) further comprises a4-hydroxybutyryl-CoA pathway. The invention further provides a methodfor producing 4-hydroxybutyrate utilizing such microbial organisms. Itis understood that such genetic modifications include, but are notlimited to, the specifically described gene additions and disruptionsdescribed in Examples XXIV-XXXIX. In the case of gene disruptions, it isunderstood that such disruptions involve disruption of an endogenousgene encoding an activity of a corresponding gene product to bedecreased.

Although the pathways immediately above are directed to 4-HB pathways,it is understood, as disclosed herein, the 4-HB is a precursor todownstream products such as 1,4-butanediol (BDO). Further, as disclosedherein, a microbial organism having a 4-HB pathway can further includeenzymes that convert 4-HB to a downstream product such as BDO, asdesired. In addition, any of the pathways described herein that produce4-HB are understood to provide a 4-HB pathway, even if additional stepsin a pathway are also disclosed since such a pathway produces 4-HB.

As used herein, the term “parent microbial organism,” when used in thecontext of a genetic modification, is understood to mean a parentorganism or strain in which the particular genetic modification has notbeen made but otherwise has the same genetic makeup. For example, if astrain of microbial organism is used to make a genetic modification thatincreases expression of a gene product, the parent strain would be thestarting strain into which the heterologous gene is introduced.Similarly, a parent microbial organism of an organism in which a geneticmodification has been made to decrease expression, such as a genedisruption or gene deletion, the parent microbial organism would havethe same genetic background except for the gene disruption or deletion.However, it is understood that a parent microbial organism can differ bymore than one genetic modification, either gene addition and/ordisruption, depending on whether a single or multiple geneticmodifications are being considered. One skilled in the art will readilyunderstand the meaning of such a parent microbial organism in that themicrobial organism is considered to be an appropriate control, asunderstood in the art, for observing the effect of one or more geneticmodifications.

In addition, it is understood by those skilled in the art that a geneticmodification such as those disclosed herein can contribute to anincrease in product yield, a decrease in by-product formation, and/orimproved characteristics of the microbial organism. Such improvedcharacteristics include, but are not limited to, improved cell growth,increased energy efficiency, increased tolerance of a range of oxygenconcentrations, particularly when manifested in a scaled up productionprocess such as in large fermentors. A useful genetic modification maynot alone, when compared to a parent microbial organism, exhibit aneffect that can readily measured under a given set of conditions.However, a useful genetic modification can be advantageous if themodification on its on under a given set of conditions and/or incombination with one or more other genetic modifications is beneficialto the growth and/or production characteristics of the microbialorganism.

It is understood that a genetic modification that increases expressionof a desired enzyme is generally carried out by introducing into themicrobial organism a heterologous nucleic acid encoding the desiredenzyme. However, it is understood, as described herein, that a metabolicmodification to increase expression can include modification of aregulatory region of an endogenous gene, such as a promoter and/orenhancer. Additionally, a genetic modification that decreases expressionof a desired enzyme will generally involve a gene disruption ordeletion, although modification of a regulatory to decrease expressioncan also be utilized, as disclosed herein.

It is further understood that any of a number of genetic modifications,as disclosed herein, can be used alone or in various combinations of oneor more of the genetic modifications disclosed herein to increase theproduction of BDO in a BDO producing microbial organism. In a particularembodiment, the microbial organism can be genetically modified toincorporate any and up to all of the genetic modifications that lead toincreased production of BDO. In a particular embodiment, the microbialorganism containing a BDO pathway can be genetically modified to expressexogenous succinyl-CoA synthetase; to express exogenousalpha-ketoglutarate decarboxylase; to express exogenous succinatesemialdehyde dehydrogenase and 4-hydroxybutyrate dehydrogenase andoptionally 4-hydroxybutyryl-CoA/acetyl-CoA transferase; to expressexogenous butyrate kinase and phosphotransbutyrylase; to expressexogenous 4-hydroxybutyryl-CoA reductase; and to express exogenous4-hydroxybutanal reductase; to express exogenous pyruvate dehydrogenase;to disrupt a gene encoding an aerobic respiratory control regulatorysystem; to express an exogenous NADH insensitive citrate synthase; andto express exogenous phosphoenolpyruvate carboxykinase. Such strains forimproved production are described in Examples XII-XIX. It is thusunderstood that, in addition to the modifications described above, suchstrains can additionally include other modifications disclosed herein.Such modifications include, but are not limited to, deletion ofendogenous lactate dehydrogenase (ldhA), alcohol dehydrogenase (adhE),and/or pyruvate formate lyase (pflB) (see Examples XII-XIX and Table28). Additional genetic modifications that can be introduced into amicrobial organism include those described in Examples XXIV-XXXX, aloneor in combination, including in combination with other geneticmodifications disclosed herein.

Additionally provided is a microbial organism in which one or more genesencoding the exogenously expressed enzymes are integrated into the fimDlocus of the host organism (see Example XVII). For example, one or moregenes encoding a BDO pathway enzyme can be integrated into the fimDlocus for increased production of BDO. Further provided is a microbialorganism expressing a non-phosphotransferase sucrose uptake system thatincreases production of BDO.

Although the genetically modified microbial organisms disclosed hereinare exemplified with microbial organisms containing particular BDOpathway enzymes, it is understood that such modifications can beincorporated into any microbial organism having a BDO, 4-HBal, 4-HBCoAand/or putrescine pathway suitable for enhanced production in thepresence of the genetic modifications of any of the pathways disclosedherein. The microbial organisms of the invention can thus have any ofthe BDO, 4-HBal, 4-HBCoA and/or putrescine pathways disclosed herein.For example, the BDO pathway can comprise 4-hydroxybutanoatedehydrogenase, succinyl-CoA synthetase, CoA-dependent succinicsemialdehyde dehydrogenase, 4-hydroxybutyrate:CoA transferase,4-butyrate kinase, phosphotransbutyrylase, alpha-ketoglutaratedecarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase or analdehyde/alcohol dehydrogenase (see FIG. 1). Alternatively, the BDOpathway can comprise 4-aminobutyrate CoA transferase, 4-aminobutyryl-CoAhydrolase, 4-aminobutyrate-CoA ligase, 4-aminobutyryl-CoA oxidoreductase(deaminating), 4-aminobutyryl-CoA transaminase, or 4-hydroxybutyryl-CoAdehydrogenase (see Table 17). Such a BDO pathway can further comprise4-hydroxybutyryl-CoA reductase (alcohol forming), 4-hydroxybutyryl-CoAreductase, or 1,4-butanediol dehydrogenase.

Additionally, the BDO pathway can comprise 4-aminobutyrate CoAtransferase, 4-aminobutyryl-CoA hydrolase, 4-aminobutyrate-CoA ligase,4-aminobutyryl-CoA reductase (alcohol forming), 4-aminobutyryl-CoAreductase, 4-aminobutan-1-ol dehydrogenase, 4-aminobutan-1-oloxidoreductase (deaminating) or 4-aminobutan-1-ol transaminase (seeTable 18). Also, the BDO pathway can comprise 4-aminobutyrate kinase,4-aminobutyraldehyde dehydrogenase (phosphorylating), 4-aminobutan-1-oldehydrogenase, 4-aminobutan-1-oloxidoreductase (deaminating),4-aminobutan-1-ol transaminase, [(4-aminobutanolyl)oxy]phosphonic acidoxidoreductase (deaminating), [(4-aminobutanolyl)oxy]phosphonic acidtransaminase, 4-hydroxybutyryl-phosphate dehydrogenase, or4-hydroxybutyraldehyde dehydrogenase (phosphorylating) (see Table 19).Such a pathway can further comprise 1,4-butanediol dehydrogenase.

The BDO pathway can also comprise alpha-ketoglutarate 5-kinase,2,5-dioxopentanoic semialdehyde dehydrogenase (phosphorylating),2,5-dioxopentanoic acid reductase, alpha-ketoglutarate CoA transferase,alpha-ketoglutaryl-CoA hydrolase, alpha-ketoglutaryl-CoA ligase,alpha-ketoglutaryl-CoA reductase, 5-hydroxy-2-oxopentanoic aciddehydrogenase, alpha-ketoglutaryl-CoA reductase (alcohol forming),5-hydroxy-2-oxopentanoic acid decarboxylase, or 5-hydroxy-2-oxopentanoicacid dehydrogenase (decarboxylation) (see Table 20). Such a BDO pathwaycan further comprise 4-hydroxybutyryl-CoA reductase (alcohol forming),4-hydroxybutyryl-CoA reductase, or 1,4-butanediol dehydrogenase.Additionally, the BDO pathway can comprise glutamate CoA transferase,glutamyl-CoA hydrolase, glutamyl-CoA ligase, glutamate 5-kinase,glutamate-5-semialdehyde dehydrogenase (phosphorylating), glutamyl-CoAreductase, glutamate-5-semialdehyde reductase, glutamyl-CoA reductase(alcohol forming), 2-amino-5-hydroxypentanoic acid oxidoreductase(deaminating), 2-amino-5-hydroxypentanoic acid transaminase,5-hydroxy-2-oxopentanoic acid decarboxylase, 5-hydroxy-2-oxopentanoicacid dehydrogenase (decarboxylation) (see Table 21). Such a BDO pathwaycan further comprise 4-hydroxybutyryl-CoA reductase (alcohol forming),4-hydroxybutyryl-CoA reductase, or 1,4-butanediol dehydrogenase.

Additionally, the BDO pathway can comprise 3-hydroxybutyryl-CoAdehydrogenase, 3-hydroxybutyryl-CoA dehydratase, vinylacetyl-CoAΔ-isomerase, or 4-hydroxybutyryl-CoA dehydratase (see Table 22). Also,the BDO pathway can comprise homoserine deaminase, homoserine CoAtransferase, homoserine-CoA hydrolase, homoserine-CoA ligase,homoserine-CoA deaminase, 4-hydroxybut-2-enoyl-CoA transferase,4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA ligase,4-hydroxybut-2-enoate reductase, 4-hydroxybutyryl-CoA transferase,4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or4-hydroxybut-2-enoyl-CoA reductase (see Table 23). Such a BDO pathwaycan further comprise 4-hydroxybutyryl-CoA reductase (alcohol forming),4-hydroxybutyryl-CoA reductase, or 1,4-butanediol dehydrogenase.

The BDO pathway can additionally comprise succinyl-CoA reductase(alcohol forming), 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoAligase, or 4-hydroxybutanal dehydrogenase (phosphorylating) (see Table15). Such a pathway can further comprise succinyl-CoA reductase,4-hydroxybutyrate dehydrogenase, 4-hydroxybutyryl-CoA transferase,4-hydroxybutyrate kinase, phosphotrans-4-hydroxybutyrylase,4-hydroxybutyryl-CoA reductase, 4-hydroxybutyryl-CoA reductase (alcoholforming), or 1,4-butanediol dehydrogenase. Also, the BDO pathway cancomprise glutamate dehydrogenase, 4-aminobutyrate oxidoreductase(deaminating), 4-aminobutyrate transaminase, glutamate decarboxylase,4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or4-hydroxybutanal dehydrogenase (phosphorylating) (see Table 16). Such aBDO pathway can further comprise alpha-ketoglutarate decarboxylase,4-hydroxybutyrate dehydrogenase, 4-hydroxybutyryl-CoA transferase,4-hydroxybutyrate kinase, phosphotrans-4-hydroxybutyrylase,4-hydroxybutyryl-CoA reductase, 4-hydroxybutyryl-CoA reductase (alcoholforming), or 1,4-butanediol dehydrogenase.

The invention additionally provides a non-naturally occurring microbialorganism, comprising a 4-hydroxybutanal pathway comprising at least oneexogenous nucleic acid encoding a 4-hydroxybutanal pathway enzymeexpressed in a sufficient amount to produce 4-hydroxybutanal, the4-hydroxybutanal pathway comprising succinyl-CoA reductase (aldehydeforming); 4-hydroxybutyrate dehydrogenase; and 4-hydroxybutyratereductase (see FIG. 58, steps A-C-D). The invention also provides anon-naturally occurring microbial organism, comprising a4-hydroxybutanal pathway comprising at least one exogenous nucleic acidencoding a 4-hydroxybutanal pathway enzyme expressed in a sufficientamount to produce 4-hydroxybutanal, the 4-hydroxybutanal pathwaycomprising alpha-ketoglutarate decarboxylase; 4-hydroxybutyratedehydrogenase; and 4-hydroxybutyrate reductase (FIG. 58, steps B-C-D).

The invention further provides a non-naturally occurring microbialorganism, comprising a 4-hydroxybutanal pathway comprising at least oneexogenous nucleic acid encoding a 4-hydroxybutanal pathway enzymeexpressed in a sufficient amount to produce 4-hydroxybutanal, the4-hydroxybutanal pathway comprising succinate reductase;4-hydroxybutyrate dehydrogenase, and 4-hydroxybutyrate reductase (seeFIG. 62, steps F-C-D). In yet another embodiment, the invention providesa non-naturally occurring microbial organism, comprising a4-hydroxybutanal pathway comprising at least one exogenous nucleic acidencoding a 4-hydroxybutanal pathway enzyme expressed in a sufficientamount to produce 4-hydroxybutanal, the 4-hydroxybutanal pathwaycomprising alpha-ketoglutarate decarboxylase, or glutamate dehydrogenaseor glutamate transaminase and glutamate decarboxylase and4-aminobutyrate dehydrogenase or 4-aminobutyrate transaminase;4-hydroxybutyrate dehydrogenase; and 4-hydroxybutyrate reductase (seeFIG. 62, steps B or ((J or K)-L-(M or N))-C-D).

The invention also provides a non-naturally occurring microbialorganism, comprising a 4-hydroxybutanal pathway comprising at least oneexogenous nucleic acid encoding a 4-hydroxybutanal pathway enzymeexpressed in a sufficient amount to produce 4-hydroxybutanal, the4-hydroxybutanal pathway comprising alpha-ketoglutarate reductase;5-hydroxy-2-oxopentanoate dehydrogenase; and 5-hydroxy-2-oxopentanoatedecarboxylase (see FIG. 62, steps X-Y-Z). In yet another embodiment, theinvention provides a non-naturally occurring microbial organism,comprising a 4-hydroxybutyryl-CoA pathway comprising at least oneexogenous nucleic acid encoding a 4-hydroxybutyryl-CoA pathway enzymeexpressed in a sufficient amount to produce 4-hydroxybutyryl-CoA, the4-hydroxybutyryl-CoA pathway comprising alpha-ketoglutarate reductase;5-hydroxy-2-oxopentanoate dehydrogenase; and 5-hydroxy-2-oxopentanoatedehydrogenase (decarboxylation) (see FIG. 62, steps X-Y-AA).

The invention additionally provides a non-naturally occurring microbialorganism, comprising a putrescine pathway comprising at least oneexogenous nucleic acid encoding a putrescine pathway enzyme expressed ina sufficient amount to produce putrescine, the putrescine pathwaycomprising succinate reductase; 4-aminobutyrate dehydrogenase or4-aminobutyrate transaminase; 4-aminobutyrate reductase; and putrescinedehydrogenase or putrescine transaminase (see FIG. 63, stepsF-M/N-C-D/E). In still another embodiment, the invention provides anon-naturally occurring microbial organism, comprising a putrescinepathway comprising at least one exogenous nucleic acid encoding aputrescine pathway enzyme expressed in a sufficient amount to produceputrescine, the putrescine pathway comprising alpha-ketoglutaratedecarboxylase; 4-aminobutyrate dehydrogenase or 4-aminobutyratetransaminase; 4-aminobutyrate reductase; and putrescine dehydrogenase orputrescine transaminase (see FIG. 63, steps B-M/N-C-D/E). The inventionadditionally provides a non-naturally occurring microbial organism,comprising a putrescine pathway comprising at least one exogenousnucleic acid encoding a putrescine pathway enzyme expressed in asufficient amount to produce putrescine, the putrescine pathwaycomprising glutamate dehydrogenase or glutamate transaminase; glutamatedecarboxylase; 4-aminobutyrate reductase; and putrescine dehydrogenaseor putrescine transaminase (see FIG. 63, steps J/K-L-C-DIE).

The invention provides in another embodiment a non-naturally occurringmicrobial organism, comprising a putrescine pathway comprising at leastone exogenous nucleic acid encoding a putrescine pathway enzymeexpressed in a sufficient amount to produce putrescine, the putrescinepathway comprising alpha-ketoglutarate reductase;5-amino-2-oxopentanoate dehydrogenase or 5-amino-2-oxopentanoatetransaminase; 5-amino-2-oxopentanoate decarboxylase; and putrescinedehydrogenase or putrescine transaminase (see FIG. 63, stepsO-P/Q-R-D/E). Also provided is a non-naturally occurring microbialorganism, comprising a putrescine pathway comprising at least oneexogenous nucleic acid encoding a putrescine pathway enzyme expressed ina sufficient amount to produce putrescine, the putrescine pathwaycomprising alpha-ketoglutarate reductase; 5-amino-2-oxopentanoatedehydrogenase or 5-amino-2-oxopentanoate transaminase; ornithinedehydrogenase or ornithine transaminase; and ornithine decarboxylase(see FIG. 63, steps O-P/Q-S/T-U).

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism having a 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine pathway, wherein the non-naturally occurring microbialorganism comprises at least one exogenous nucleic acid encoding anenzyme or protein that converts a substrate of any of the pathwaysdisclosed herein (see, for example, the Examples and FIGS. 1, 8-13, 58,62 and 63). In an exemplary embodiment for producing BDO, the microbialorganism can convert a substrate to a product selected from the groupconsisting of succinate to succinyl-CoA; succinyl-CoA to succinicsemialdehyde; succinic semialdehyde to 4-hydroxybutrate;4-hydroxybutyrate to 4-hydroxybutyryl-phosphate;4-hydroxybutyryl-phosphate to 4-hydroxtbutyryl-CoA; 4-hydroxybutyryl-CoAto 4-hydroxybutanal; and 4-hydroxybutanal to 1,4-butanediol. In apathway for producing 4-HBal, a microbial organism can convert, forexample, succinate to succinic semialdehyde; succinic semialdehyde to4-hydroxybutyrate; and 4-hydroxybutyrate to 4-hydroxybutanal. Such anorganism can additionally include activity to convert 4-hydroxybutanalto 1,4-butanediol in order to produce BDO. Yet another pathway forproducing 4-HBal can be, for example, alpha-ketoglutarate to succinicsemialdehyde; succinic semialdehyde to 4-hydroxybutyrate; and4-hydroxybutyrate to 4-hydroxybutanal. An alternative pathway forproducing 4-HBal can be, for example, alpha-ketoglutarate to2,5-dioxopentanoic acid; 2,5-dioxopentanoic acid to5-hydroxy-2-oxopentanooic acid; and 5-hydroxy-2-oxopentanoic acid to4-hydroxybutanal. An exemplary 4-hydroxybutyryl-CoA pathway can be, forexample, alpha-ketoglutarate to 2,5-dioxopentanoic acid;2,5-dioxopentanoic acid to 5-hydroxy-2-oxopentanoic acid; and5-hydroxy-2-oxopentanoic acid to 4-hydroxybutyryl-CoA. An exemplaryputrescine pathway can be, for example, succinate to succinyl-CoA;succinyl-CoA to succinic semialdehyde; succinic semialdehyde to4-aminobutyrate; 4-aminobutyrate to 4-aminobutanal; and 4-aminobutanalto putrescine. An alternative putrescine pathway can be, for example,succinate to succinic semialdehyde; succinic semialdehyde to4-aminobutyrate; 4-aminobutyrate to 4-aminobutanal; and 4-aminobutanalto putrescine. One skilled in the art will understand that these aremerely exemplary and that any of the substrate-product pairs disclosedherein suitable to produce a desired product and for which anappropriate activity is available for the conversion can be readilydetermined by one skilled in the art based on the teachings herein.Thus, the invention provides a non-naturally occurring microbialorganism containing at least one exogenous nucleic acid encoding anenzyme or protein, where the enzyme or protein converts the substratesand products of a pathway (see FIGS. 1, 8-13, 58, 62 and 63).

While generally described herein as a microbial organism that contains a4-HB, 4-HBal, 4-HBCoA, BDO or putrescine pathway, it is understood thatthe invention additionally provides a non-naturally occurring microbialorganism comprising at least one exogenous nucleic acid encoding a 4-HB,4-HBal, 4-HBCoA, BDO or putrescine pathway enzyme or protein expressedin a sufficient amount to produce an intermediate of a 4-HB, 4-HBal,4-HBCoA, BDO or putrescine pathway. For example, as disclosed herein,4-HB, 4-HBal, 4-HBCoA, BDO and putrescine pathways are exemplified inFIGS. 1, 8-13, 58, 62 and 63. Therefore, in addition to a microbialorganism containing, for example, a BDO pathway that produces BDO, theinvention additionally provides a non-naturally occurring microbialorganism comprising at least one exogenous nucleic acid encoding a BDOpathway enzyme, where the microbial organism produces a BDO pathwayintermediate as a product rather than an intermediate of the pathway. Inone exemplary embodiment as shown in FIG. 62, for example, the inventionprovides a microbial organism that produces succinyl-CoA, succinicsemialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl-phosphate,4-hydroxybutyryl-CoA, or 4-hydroxybutanal as a product rather than anintermediate. Another exemplary embodiment includes, for example, amicrobial organism that produces alpha-ketoglutarate, 2,5-dioxopentanoicacid, 5-hydroxy-2-oxopentanoic acid, or 4-hydroxybutanal as a productrather than an intermediate. An exemplary embodiment in a putrescinepathway includes, for example, a microbial organism that producesglutamate, 4-aminobutyrate, or 4-aminobutanal as a product rather thanan intermediate. An alternative embodiment in a putrescine pathway canbe, for example, a microbial organism that produces 2,5-dioxopentanoate,5-amino-2-oxopentanoate, or ornithine as a product rather than anintermediate.

It is understood that any of the pathways disclosed herein, as describedin the Examples and exemplified in the Figures, including the pathwaysof FIGS. 1, 8-13, 58, 62 and 63, can be utilized to generate anon-naturally occurring microbial organism that produces any pathwayintermediate or product, as desired. As disclosed herein, such amicrobial organism that produces an intermediate can be used incombination with another microbial organism expressing downstreampathway enzymes to produce a desired product. However, it is understoodthat a non-naturally occurring microbial organism that produces a 4-HB,4-HBal, 4-HBCoA, BDO or putrescine pathway intermediate can be utilizedto produce the intermediate as a desired product.

The invention is described herein with general reference to themetabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing the referenced metabolic reaction,reactant or product. Unless otherwise expressly stated herein, thoseskilled in the art will understand that reference to a reaction alsoconstitutes reference to the reactants and products of the reaction.Similarly, unless otherwise expressly stated herein, reference to areactant or product also references the reaction and that reference toany of these metabolic constitutes also references the gene or genesencoding the enzymes that catalyze the referenced reaction, reactant orproduct. Likewise, given the well known fields of metabolicbiochemistry, enzymology and genomics, reference herein to a gene orencoding nucleic acid also constitutes a reference to the correspondingencoded enzyme and the reaction it catalyzes as well as the reactantsand products of the reaction.

As disclosed herein, the product 4-hydroxybutyrate, as well as otherintermediates, are carboxylic acids, which can occur in various ionizedforms, including fully protonated, partially protonated, and fullydeprotonated forms. Accordingly, the suffix “-ate,” or the acid form,can be used interchangeably to describe both the free acid form as wellas any deprotonated form, in particular since the ionized form is knownto depend on the pH in which the compound is found. It is understoodthat carboxylate products or intermediates includes ester forms ofcarboxylate products or pathway intermediates, such as O-carboxylate andS-carboxylate esters. O- and S-carboxylates can include lower alkyl,that is C1 to C6, branched or straight chain carboxylates. Some such O-or S-carboxylates include, without limitation, methyl, ethyl, n-propyl,n-butyl, i-propyl, sec-butyl, and tert-butyl, pentyl, hexyl O- orS-carboxylates, any of which can further possess an unsaturation,providing for example, propenyl, butenyl, pentyl, and hexenyl O- orS-carboxylates. O-carboxylates can be the product of a biosyntheticpathway. Exemplary O-carboxylates accessed via biosynthetic pathways caninclude, without limitation, methyl 4-hydroxybutyrate, ethyl4-hydroxybutyrate, and n-propyl 4-hydroxybutyrate. Otherbiosynthetically accessible O-carboxylates can include medium to longchain groups, that is C7-C22, O-carboxylate esters derived from fattyalcohols, such heptyl, octyl, nonyl, decyl, undecyl, lauryl, tridecyl,myristyl, pentadecyl, cetyl, palmitolyl, heptadecyl, stearyl, nonadecyl,arachidyl, heneicosyl, and behenyl alcohols, any one of which can beoptionally branched and/or contain unsaturations. O-carboxylate esterscan also be accessed via a biochemical or chemical process, such asesterification of a free carboxylic acid product or transesterificationof an O- or S-carboxylate. S-carboxylates are exemplified by CoAS-esters, cysteinyl S-esters, alkylthioesters, and various aryl andheteroaryl thioesters.

The production of 4-HB via biosynthetic modes using the microbialorganisms of the invention is particularly useful because it can producemonomeric 4-HB. The non-naturally occurring microbial organisms of theinvention and their biosynthesis of 4-HB and BDO family compounds alsois particularly useful because the 4-HB product can be (1) secreted; (2)can be devoid of any derivatizations such as Coenzyme A; (3) avoidsthermodynamic changes during biosynthesis; (4) allows directbiosynthesis of BDO, and (5) allows for the spontaneous chemicalconversion of 4-HB to γ-butyrolactone (GBL) in acidic pH medium. Thislatter characteristic also is particularly useful for efficient chemicalsynthesis or biosynthesis of BDO family compounds such as 1,4-butanedioland/or tetrahydrofuran (THF), for example.

Microbial organisms generally lack the capacity to synthesize 4-HB andtherefore any of the compounds disclosed herein to be within the1,4-butanediol family of compounds or known by those in the art to bewithin the 1,4-butanediol family of compounds. Moreover, organismshaving all of the requisite metabolic enzymatic capabilities are notknown to produce 4-HB from the enzymes described and biochemicalpathways exemplified herein. Rather, with the possible exception of afew anaerobic microorganisms described further below, the microorganismshaving the enzymatic capability to use 4-HB as a substrate to produce,for example, succinate. In contrast, the non-naturally occurringmicrobial organisms of the invention can generate 4-HB, 4-HBal, 4-HBCoA,BDO and/or putrescine as a product. As described above, the biosynthesisof 4-HB in its monomeric form is not only particularly useful inchemical synthesis of BDO family of compounds, it also allows for thefurther biosynthesis of BDO family compounds and avoids altogetherchemical synthesis procedures.

The non-naturally occurring microbial organisms of the invention thatcan produce 4-HB, 4-HBal, 4-HBCoA, BDO and/or putrescine are produced byensuring that a host microbial organism includes functional capabilitiesfor the complete biochemical synthesis of at least one 4-HB, 4-HBal,4-HBCoA, BDO and/or putrscine biosynthetic pathway of the invention.Ensuring at least one requisite 4-HB, 4-HBal, 4-HBCoA or BDObiosynthetic pathway confers 4-HB biosynthesis capability onto the hostmicrobial organism.

Several 4-HB biosynthetic pathways are exemplified herein and shown forpurposes of illustration in FIG. 1. Additional 4-HB and BDO pathways aredescribed in FIGS. 8-13. One 4-HB biosynthetic pathway includes thebiosynthesis of 4-HB from succinate (the succinate pathway). The enzymesparticipating in this 4-HB pathway include CoA-independent succinicsemialdehyde dehydrogenase and 4-hydroxybutanoate dehydrogenase. In thispathway, CoA-independent succinic semialdehyde dehydrogenase catalyzesthe reverse reaction to the arrow shown in FIG. 1. Another 4-HBbiosynthetic pathway includes the biosynthesis from succinate throughsuccinyl-CoA (the succinyl-CoA pathway). The enzymes participating inthis 4-HB pathway include succinyl-CoA synthetase, CoA-dependentsuccinic semialdehyde dehydrogenase and 4-hydroxybutanoatedehydrogenase. Three other 4-HB biosynthetic pathways include thebiosynthesis of 4-HB from α-ketoglutarate (the α-ketoglutaratepathways). Hence, a third 4-HB biosynthetic pathway is the biosynthesisof succinic semialdehyde through glutamate: succinic semialdehydetransaminase, glutamate decarboxylase and 4-hydroxybutanoatedehydrogenase. A fourth 4-HB biosynthetic pathway also includes thebiosynthesis of 4-HB from α-ketoglutarate, but utilizes α-ketoglutaratedecarboxylase to catalyze succinic semialdehyde synthesis.4-hydroxybutanoate dehydrogenase catalyzes the conversion of succinicsemialdehyde to 4-HB. A fifth 4-HB biosynthetic pathway includes thebiosynthesis from α-ketoglutarate through succinyl-CoA and utilizesα-ketoglutarate dehydrogenase to produce succinyl-CoA, which funnelsinto the succinyl-CoA pathway described above. Each of these 4-HBbiosynthetic pathways, their substrates, reactants and products aredescribed further below in the Examples. As described herein, 4-HB canfurther be biosynthetically converted to BDO by inclusion of appropriateenzymes to produce BDO (see Example). Thus, it is understood that a 4-HBpathway can be used with enzymes for converting 4-HB to BDO to generatea BDO pathway.

The non-naturally occurring microbial organisms of the invention can beproduced by introducing expressible nucleic acids encoding one or moreof the enzymes participating in one or more 4-HB, 4-HBal, 4-HBCoA, BDOor putrescine biosynthetic pathways. Depending on the host microbialorganism chosen for biosynthesis, nucleic acids for some or all of aparticular 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine biosynthetic pathwaycan be expressed. For example, if a chosen host is deficient in one ormore enzymes in a desired biosynthetic pathway, for example, thesuccinate to 4-HB pathway, then expressible nucleic acids for thedeficient enzyme(s), for example, both CoA-independent succinicsemialdehyde dehydrogenase and 4-hydroxybutanoate dehydrogenase in thisexample, are introduced into the host for subsequent exogenousexpression. Alternatively, if the chosen host exhibits endogenousexpression of some pathway enzymes, but is deficient in others, then anencoding nucleic acid is needed for the deficient enzyme(s) to achieve4-HB, 4-HBal, 4-HBCoA, BDO or putrescine biosynthesis. For example, ifthe chosen host exhibites endogenous CoA-independent succinicsemialdehyde dehydrogenase, but is deficient in 4-hydroxybutanoatedehydrogenase, then an encoding nucleic acid is needed for this enzymeto achieve 4-HB biosynthesis. Thus, a non-naturally occurring microbialorganism of the invention can be produced by introducing exogenousenzyme or protein activities to obtain a desired biosynthetic pathway ora desired biosynthetic pathway can be obtained by introducing one ormore exogenous enzyme or protein activities that, together with one ormore endogenous enzymes or proteins, produces a desired product such as4-HB, 4-HBal, 4-HBCoA, BDO and/or putrescine.

In like fashion, where 4-HB biosynthesis is selected to occur throughthe succinate to succinyl-CoA pathway (the succinyl-CoA pathway),encoding nucleic acids for host deficiencies in the enzymes succinyl-CoAsynthetase, CoA-dependent succinic semialdehyde dehydrogenase and/or4-hydroxybutanoate dehydrogenase are to be exogenously expressed in therecipient host. Selection of 4-HB biosynthesis through theα-ketoglutarate to succinic semialdehyde pathway (the α-ketoglutaratepathway) can utilize exogenous expression for host deficiencies in oneor more of the enzymes for glutamate:succinic semialdehyde transaminase,glutamate decarboxylase and/or 4-hydroxybutanoate dehydrogenase, orα-ketoglutarate decarboxylase and 4-hydroxybutanoate dehydrogenase. Oneskilled in the art can readily determine pathway enzymes for productionof 4-HB or BDO, as disclosed herein.

Depending on the 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine biosyntheticpathway constituents of a selected host microbial organism, thenon-naturally occurring microbial organisms of the invention willinclude at least one exogenously expressed 4-HB, 4-HB, 4-HBCoA, BDO orputrescine pathway-encoding nucleic acid and up to all encoding nucleicacids for one or more 4-HB or BDO biosynthetic pathways. For example,4-HB, 4-HBal, 4-HBCoA, BDO or putrescine biosynthesis can be establishedin a host deficient in a pathway enzyme or protein through exogenousexpression of the corresponding encoding nucleic acid. In a hostdeficient in all enzymes or proteins of a 4-HB, 4-HB, 4-HBCoA, BDO orputrescine pathway, exogenous expression of all enzyme or proteins inthe pathway can be included, although it is understood that all enzymesor proteins of a pathway can be expressed even if the host contains atleast one of the pathway enzymes or proteins. If desired, exogenousexpression of all enzymes or proteins in a pathway for production of4-HB, 4-HB, 4-HBCoA, BDO or putrescine can be included. For example,4-HB biosynthesis can be established from all five pathways in a hostdeficient in 4-hydroxybutanoate dehydrogenase through exogenousexpression of a 4-hydroxybutanoate dehydrogenase encoding nucleic acid.In contrast, 4-HB biosynthesis can be established from all five pathwaysin a host deficient in all eight enzymes through exogenous expression ofall eight of CoA-independent succinic semialdehyde dehydrogenase,succinyl-CoA synthetase, CoA-dependent succinic semialdehydedehydrogenase, glutamate: succinic semialdehyde transaminase, glutamatedecarboxylase, α-ketoglutarate decarboxylase, α-ketoglutaratedehydrogenase and 4-hydroxybutanoate dehydrogenase.

Given the teachings and guidance provided herein, those skilled in theart will understand that the number of encoding nucleic acids tointroduce in an expressible form will, at least, parallel 4-HB, 4-HBal,4-HBCoA, BDO or putrescine pathway deficiencies of the selected hostmicrobial organism. Therefore, a non-naturally occurring microbialorganism of the invention can have one, two, three, four, five, six,seven, eight or up to all nucleic acids encoding the enzymes disclosedherein constituting one or more 4-HB, 4-HBal, 4-HBCoA, BDO or putrescinebiosynthetic pathways. In some embodiments, the non-naturally occurringmicrobial organisms also can include other genetic modifications thatfacilitate or optimize 4-HB, 4-HBal, 4-HBCoA, BDO or putrescinebiosynthesis or that confer other useful functions onto the hostmicrobial organism. One such other functionality can include, forexample, augmentation of the synthesis of one or more of 4-HB pathwayprecursors such as succinate, succinyl-CoA, α-ketoglutarate,4-aminobutyrate, glutamate, acetoacetyl-CoA, and/or homoserine.

Generally, a host microbial organism is selected such that it producesthe precursor of a 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine pathway,either as a naturally produced molecule or as an engineered product thateither provides de novo production of a desired precursor or increasedproduction of a precursor naturally produced by the host microbialorganism. For example, succinyl-CoA, α-ketoglutarate, 4-aminobutyrate,glutamate, acetoacetyl-CoA, and homoserine are produced naturally in ahost organism such as E. coli. A host organism can be engineered toincrease production of a precursor, as disclosed herein. In addition, amicrobial organism that has been engineered to produce a desiredprecursor can be used as a host organism and further engineered toexpress enzymes or proteins of a 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine pathway.

In some embodiments, a non-naturally occurring microbial organism of theinvention is generated from a host that contains the enzymaticcapability to synthesize 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine. Inthis specific embodiment it can be useful to increase the synthesis oraccumulation of a 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine pathwayproduct to, for example, drive 4-HB, 4-HBal, 4-HBCoA, BDO or putrescinepathway reactions toward 4-HB, 4-HBal, 4-HBCoA, BDO or putrescineproduction. Increased synthesis or accumulation can be accomplished by,for example, overexpression of nucleic acids encoding one or more of the4-HB, 4-HBal, 4-HBCoA, BDO or putrescine pathway enzymes disclosedherein. Over expression of the 4-HB, 4-HBal, 4-HBCoA, BDO or putrescinepathway enzyme or enzymes can occur, for example, through exogenousexpression of the endogenous gene or genes, or through exogenousexpression of the heterologous gene or genes. Therefore, naturallyoccurring organisms can be readily generated to be non-naturally 4-HB,4-HBal, 4-HBCoA, BDO or putrescine producing microbial organisms of theinvention through overexpression of one, two, three, four, five, six andso forth up to all nucleic acids encoding 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine biosynthetic pathway enzymes. In addition, a non-naturallyoccurring organism can be generated by mutagenesis of an endogenous genethat results in an increase in activity of an enzyme in the 4-HB,4-HBal, 4-HBCoA, BDO or putrescine biosynthetic pathway.

In particularly useful embodiments, exogenous expression of the encodingnucleic acids is employed. Exogenous expression confers the ability tocustom tailor the expression and/or regulatory elements to the host andapplication to achieve a desired expression level that is controlled bythe user. However, endogenous expression also can be utilized in otherembodiments such as by removing a negative regulatory effector orinduction of the gene's promoter when linked to an inducible promoter orother regulatory element. Thus, an endogenous gene having a naturallyoccurring inducible promoter can be up-regulated by providing theappropriate inducing agent, or the regulatory region of an endogenousgene can be engineered to incorporate an inducible regulatory element,thereby allowing the regulation of increased expression of an endogenousgene at a desired time. Similarly, an inducible promoter can be includedas a regulatory element for an exogenous gene introduced into anon-naturally occurring microbial organism (see Examples).

“Exogenous” as it is used herein is intended to mean that the referencedmolecule or the referenced activity is introduced into the hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid of theinvention can utilize either or both a heterologous or homologousencoding nucleic acid.

It is understood that when more than one exogenous nucleic acid isincluded in a microbial organism that the more than one exogenousnucleic acids refers to the referenced encoding nucleic acid orbiosynthetic activity, as discussed above. It is further understood, asdisclosed herein, that such more than one exogenous nucleic acids can beintroduced into the host microbial organism on separate nucleic acidmolecules, on polycistronic nucleic acid molecules, or a combinationthereof, and still be considered as more than one exogenous nucleicacid. For example, as disclosed herein a microbial organism can beengineered to express two or more exogenous nucleic acids encoding adesired pathway enzyme or protein. In the case where two exogenousnucleic acids encoding a desired activity are introduced into a hostmicrobial organism, it is understood that the two exogenous nucleicacids can be introduced as a single nucleic acid, for example, on asingle plasmid, on separate plasmids, can be integrated into the hostchromosome at a single site or multiple sites, and still be consideredas two exogenous nucleic acids. Similarly, it is understood that morethan two exogenous nucleic acids can be introduced into a host organismin any desired combination, for example, on a single plasmid, onseparate plasmids, can be integrated into the host chromosome at asingle site or multiple sites, and still be considered as two or moreexogenous nucleic acids, for example three exogenous nucleic acids.Thus, the number of referenced exogenous nucleic acids or biosyntheticactivities refers to the number of encoding nucleic acids or the numberof biosynthetic activities, not the number of separate nucleic acidsintroduced into the host organism.

As used herein, the term “gene disruption,” or grammatical equivalentsthereof, is intended to mean a genetic alteration that renders theencoded gene product inactive. The genetic alteration can be, forexample, deletion of the entire gene, deletion of a regulatory sequencerequired for transcription or translation, deletion of a portion of thegene which results in a truncated gene product, or by any of variousmutation strategies that inactivate the encoded gene product. Oneparticularly useful method of gene disruption is complete gene deletionbecause it reduces or eliminates the occurrence of genetic reversions inthe non-naturally occurring microorganisms of the invention.

As used herein, the term “growth-coupled” when used in reference to theproduction of a biochemical product is intended to mean that thebiosynthesis of the referenced biochemical product is produced duringthe growth phase of a microorganism. In a particular embodiment, thegrowth-coupled production can optionally be obligatory, meaning that thebiosynthesis of the referenced biochemical is an obligatory productproduced during the growth phase of a microorganism.

Sources of encoding nucleic acids for a 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine pathway enzyme can include, for example, any species wherethe encoded gene product is capable of catalyzing the referencedreaction. Such species include both prokaryotic and eukaryotic organismsincluding, but not limited to, bacteria, including archaea andeubacteria, and eukaryotes, including yeast, plant, insect, animal, andmammal, including human. Exemplary species for such sources include, forexample, Escherichia coli, Saccharomyces cerevisiae, Saccharomyceskluyveri, Clostridium kluyveri, Clostridium acetobutylicum, Clostridiumbeijerinckii, Clostridium saccharoperbutylacetonicum, Clostridiumperfringens, Clostridium difficile, Clostridium botulinum, Clostridiumtyrobutyricum, Clostridium tetanomorphum, Clostridium tetani,Clostridium propionicum, Clostridium aminobutyricum, Clostridiumsubterminale, Clostridium sticklandii, Ralstonia eutropha, Mycobacteriumbovis, Mycobacterium tuberculosis, Porphyromonas gingivalis, Arabidopsisthaliana, Thermus thermophilus, Pseudomonas species, includingPseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri,Pseudomonas fluorescens, Homo sapiens, Oryctolagus cuniculus,Rhodobacter spaeroides, Thermoanaerobacter brockii, Metallosphaerasedula, Leuconostoc mesenteroides, Chloroflexus aurantiacus, Roseiflexuscastenholzii, Erythrobacter, Simmondsia chinensis, Acinetobacterspecies, including Acinetobacter calcoaceticus and Acinetobacter baylyi,Porphyromonas gingivalis, Sulfolobus tokodaii, Sulfolobus solfataricus,Sulfolobus acidocaldarius, Bacillus subtilis, Bacillus cereus, Bacillusmegaterium, Bacillus brevis, Bacillus pumilus, Rattus norvegicus,Klebsiella pneumonia, Klebsiella oxytoca, Euglena gracilis, Treponemadenticola, Moorella thermoacetica, Thermotoga maritima, Halobacteriumsalinarum, Geobacillus stearothermophilus, Aeropyrum pernix, Sus scrofa,Caenorhabditis elegans, Corynebacterium glutamicum, Acidaminococcusfermentans, Lactococcus lactis, Lactobacillus plantarum, Streptococcusthermophilus, Enterobacter aerogenes, Candida, Aspergillus terreus,Pedicoccus pentosaceus, Zymomonas mobilus, Acetobacter pasteurians,Kluyveromyces lactis, Eubacterium barkeri, Bacteroides capillosus,Anaerotruncus colihominis, Natranaerobius thermophilusm, Campylobacterjejuni, Haemophilus influenzae, Serratia marcescens, Citrobacteramalonaticus, Myxococcus xanthus, Fusobacterium nuleatum, Penicilliumchrysogenum, marine gamma proteobacterium, butyrate-producing bacterium,Nocardia iowensis, Nocardia farcinica, Streptomyces griseus,Schizosaccharomyces pombe, Geobacillus thermoglucosidasius, Salmonellatyphimurium, Vibrio cholera, Heliobacter pylori, Nicotiana tabacum,Oryza sativa, Haloferax mediterranei, Agrobacterium tumefaciens,Achromobacter denitrificans, Fusobacterium nucleatum, Streptomycesclavuligenus, Acinetobacter baumanii, Mus musculus, Lachancea kluyveri,Trichomonas vaginalis, Trypanosoma brucei, Pseudomonas stutzeri,Bradyrhizobium japonicum, Mesorhizobium loti, Bos taurus, Nicotianaglutinosa, Vibrio vulnificus, Selenomonas ruminantium, Vibrioparahaemolyticus, Archaeoglobus fulgidus, Haloarcula marismortui,Pyrobaculum aerophilum, Mycobacterium smegmatis MC2 155, Mycobacteriumavium subsp. paratuberculosis K-10, Mycobacterium marinum M,Tsukamurella paurometabola DSM 20162, Cyanobium PCC7001, Dictyosteliumdiscoideum AX4, and others disclosed herein (see Examples). For example,microbial organisms having 4-HB, 4-HBal, 4-HBCoA, BDO or putrescinebiosynthetic production are exemplified herein with reference to E. coliand yeast hosts. However, with the complete genome sequence availablefor now more than 550 species (with more than half of these available onpublic databases such as the NCBI), including 395 microorganism genomesand a variety of yeast, fungi, plant, and mammalian genomes, theidentification of genes encoding the requisite 4-HB, 4-HBal, 4-HBCoA,BDO or putrescine biosynthetic activity for one or more genes in relatedor distant species, including for example, homologues, orthologs,paralogs and nonorthologous gene displacements of known genes, and theinterchange of genetic alterations between organisms is routine and wellknown in the art. Accordingly, the metabolic alterations allowingbiosynthesis of 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine and othercompounds of the invention described herein with reference to aparticular organism such as E. coli or yeast can be readily applied toother microorganisms, including prokaryotic and eukaryotic organismsalike. Given the teachings and guidance provided herein, those skilledin the art will know that a metabolic alteration exemplified in oneorganism can be applied equally to other organisms.

In some instances, such as when an alternative 4-HB, 4-HBal, BDO orputrescine biosynthetic pathway exists in an unrelated species, 4-HB,4-HBal, BDO or putrescine biosynthesis can be conferred onto the hostspecies by, for example, exogenous expression of a paralog or paralogsfrom the unrelated species that catalyzes a similar, yet non-identicalmetabolic reaction to replace the referenced reaction. Because certaindifferences among metabolic networks exist between different organisms,those skilled in the art will understand that the actual gene usagebetween different organisms may differ. However, given the teachings andguidance provided herein, those skilled in the art also will understandthat the teachings and methods of the invention can be applied to allmicrobial organisms using the cognate metabolic alterations to thoseexemplified herein to construct a microbial organism in a species ofinterest that will synthesize 4-HB, such as monomeric 4-HB, 4-HBal, BDOor putrescine.

Host microbial organisms can be selected from, and the non-naturallyoccurring microbial organisms generated in, for example, bacteria,yeast, fungus or any of a variety of other microorganisms applicable tofermentation processes. Exemplary bacteria include species selected fromEscherichia coli, Klebsiella oxytoca, Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes, Mannheimiasucciniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacteriumglutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcuslactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridiumacetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.Exemplary yeasts or fungi include species selected from Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis,Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichiapastoris, Rhizopus arrhizus, Rhizobus oryzae, and the like. E. coli is aparticularly useful host organisms since it is a well characterizedmicrobial organism suitable for genetic engineering. Other particularlyuseful host organisms include yeast such as Saccharomyces cerevisiae. Itis understood that any suitable microbial host organism can be used tointroduce metabolic and/or genetic modifications to produce a desiredproduct.

Methods for constructing and testing the expression levels of anon-naturally occurring 4-HB-, 4-HBal-, 4-HBCoA-, BDO-, orputrescine-producing host can be performed, for example, by recombinantand detection methods well known in the art. Such methods can be founddescribed in, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York(2001); Ausubel et al., Current Protocols in Molecular Biology, JohnWiley and Sons, Baltimore, Md. (1999). 4-HB and GBL can be separated by,for example, HPLC using a Spherisorb 5 ODS 1 column and a mobile phaseof 70% 10 mM phosphate buffer (pH=7) and 30% methanol, and detectedusing a UV detector at 215 nm (Hennessy et al. 2004, J. Forensic Sci.46(6):1-9). BDO is detected by gas chromatography or by HPLC andrefractive index detector using an Aminex HPX-87H column and a mobilephase of 0.5 mM sulfuric acid (Gonzalez-Pajuelo et al., Met. Eng.7:329-336 (2005)).

Exogenous nucleic acid sequences involved in a pathway for production of4-HB, 4-HBal, 4-HBCoA, BDO or putrescine can be introduced stably ortransiently into a host cell using techniques well known in the artincluding, but not limited to, conjugation, electroporation, chemicaltransformation, transduction, transfection, and ultrasoundtransformation. For exogenous expression in E. coli or other prokaryoticcells, some nucleic acid sequences in the genes or cDNAs of eukaryoticnucleic acids can encode targeting signals such as an N-terminalmitochondrial or other targeting signal, which can be removed beforetransformation into prokaryotic host cells, if desired. For example,removal of a mitochondrial leader sequence led to increased expressionin E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)).For exogenous expression in yeast or other eukaryotic cells, genes canbe expressed in the cytosol without the addition of leader sequence, orcan be targeted to mitochondrion or other organelles, or targeted forsecretion, by the addition of a suitable targeting sequence such as amitochondrial targeting or secretion signal suitable for the host cells.Thus, it is understood that appropriate modifications to a nucleic acidsequence to remove or include a targeting sequence can be incorporatedinto an exogenous nucleic acid sequence to impart desirable properties.Furthermore, genes can be subjected to codon optimization withtechniques well known in the art to achieve optimized expression of theproteins.

An expression vector or vectors can be constructed to harbor one or more4-HB, 4-HBal, 4-HBCoA, BDO or putrescine biosynthetic pathway and/or oneor more biosynthetic encoding nucleic acids as exemplified hereinoperably linked to expression control sequences functional in the hostorganism. Expression vectors applicable for use in the microbial hostorganisms of the invention include, for example, plasmids, phagevectors, viral vectors, episomes and artificial chromosomes, includingvectors and selection sequences or markers operable for stableintegration into a host chromosome. Additionally, the expression vectorscan include one or more selectable marker genes and appropriateexpression control sequences. Selectable marker genes also can beincluded that, for example, provide resistance to antibiotics or toxins,complement auxotrophic deficiencies, or supply critical nutrients not inthe culture media. Expression control sequences can include constitutiveand inducible promoters, transcription enhancers, transcriptionterminators, and the like which are well known in the art. When two ormore exogenous encoding nucleic acids are to be co-expressed, bothnucleic acids can be inserted, for example, into a single expressionvector or in separate expression vectors. For single vector expression,the encoding nucleic acids can be operationally linked to one commonexpression control sequence or linked to different expression controlsequences, such as one inducible promoter and one constitutive promoter.The transformation of exogenous nucleic acid sequences involved in ametabolic or synthetic pathway can be confirmed using methods well knownin the art. Such methods include, for example, nucleic acid analysissuch as Northern blots or polymerase chain reaction (PCR) amplificationof mRNA, or immunoblotting for expression of gene products, or othersuitable analytical methods to test the expression of an introducednucleic acid sequence or its corresponding gene product. It isunderstood by those skilled in the art that the exogenous nucleic acidis expressed in a sufficient amount to produce the desired product, andit is further understood that expression levels can be optimized toobtain sufficient expression using methods well known in the art and asdisclosed herein.

The non-naturally occurring microbial organisms of the invention areconstructed using methods well known in the art as exemplified herein toexogenously express at least one nucleic acid encoding a 4-HB, 4-HBal,4-HBCoA, BDO or putrescine pathway enzyme in sufficient amounts toproduce 4-HB, such as monomeric 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine. It is understood that the microbial organisms of theinvention are cultured under conditions sufficient to produce 4-HB,4-HBal, 4-HBCoA, BDO or putrescine. Exemplary levels of expression for4-HB, 4-HBal, 4-HBCoA, BDO or putrescine enzymes in each pathway aredescribed further below in the Examples. Following the teachings andguidance provided herein, the non-naturally occurring microbialorganisms of the invention can achieve biosynthesis of 4-HB, such asmonomeric 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine resulting inintracellular concentrations between about 0.1-200 mM or more, forexample, 0.1-25 mM or more. Generally, the intracellular concentrationof 4-HB, such as monomeric 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine isbetween about 3-150 mM or more, particularly about 5-125 mM or more, andmore particularly between about 8-100 mM, for example, about 3-20 mM,particularly between about 5-15 mM and more particularly between about8-12 mM, including about 10 mM, 20 mM, 50 mM, 80 mM or more.Intracellular concentrations between and above each of these exemplaryranges also can be achieved from the non-naturally occurring microbialorganisms of the invention. In particular embodiments, the microbialorganisms of the invention, particularly strains such as those disclosedherein (see Examples XII-XIX and Table 28), can provide improvedproduction of a desired product such as 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine by increasing the production of 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine and/or decreasing undesirable byproducts. Such productionlevels include, but are not limited to, those disclosed herein andincluding from about 1 gram to about 25 grams per liter, for exampleabout 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or even higher amounts of product per liter.

In addition to the culturing and fermentation conditions disclosedherein, growth condition for achieving biosynthesis of BDO, 4-HB,4-HBCoA, 4-HBal and/or putrescine can include the addition of anosmoprotectant to the culturing conditions. In certain embodiments, thenon-naturally occurring microbial organisms of the invention can besustained, cultured or fermented as described herein in the presence ofan osmoprotectant. Briefly, an osmoprotectant refers to a compound thatacts as an osmolyte and helps a microbial organism as described hereinsurvive osmotic stress. Osmoprotectants include, but are not limited to,betaines, amino acids, and the sugar trehalose. Non-limiting examples ofsuch are glycine betaine, praline betaine, dimethylthetin,dimethylslfonioproprionate, 3-dimethylsulfonio-2-methylproprionate,pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine andectoine. In one aspect, the osmoprotectant is glycine betaine. It isunderstood to one of ordinary skill in the art that the amount and typeof osmoprotectant suitable for protecting a microbial organism describedherein from osmotic stress will depend on the microbial organism used.The amount of osmoprotectant in the culturing conditions can be, forexample, no more than about 0.1 mM, no more than about 0.5 mM, no morethan about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM,no more than about 2.5 mM, no more than about 3.0 mM, no more than about5.0 mM, no more than about 7.0 mM, no more than about 10 mM, no morethan about 50 mM, no more than about 100 mM or no more than about 500mM.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described herein and are described, for example, in U.S. publication2009/0047719, filed Aug. 10, 2007. Any of these conditions can beemployed with the non-naturally occurring microbial organisms as well asother anaerobic conditions well known in the art. Under such anaerobicconditions or substantially anaerobic, the 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine producers can synthesize 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine at intracellular concentrations of 5-10 mM or more as well asall other concentrations exemplified herein. It is understood that, eventhough the above description refers to intracellular concentrations,4-HB, 4-HBal, 4-HBCoA, BDO or putrescine producing microbial organismscan produce 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine intracellularlyand/or secrete the product into the culture medium.

In some embodiments, the carbon feedstock and other cellular uptakesources such as phosphate, ammonia, sulfate, chloride and other halogenscan be chosen to alter the isotopic distribution of the atoms present in4-HB, 4-HBal, 4-HBCoA, BDO or putrescine or any 4-HB, 4-HBal, 4-HBCoA,BDO or putrescine pathway intermediate. The various carbon feedstock andother uptake sources enumerated above will be referred to herein,collectively, as “uptake sources.” Uptake sources can provide isotopicenrichment for any atom present in the product 4-HB, 4-HBal, 4-HBCoA,BDO or putrescine or 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine pathwayintermediate including any 4-HB, 4-HBal, 4-HBCoA, BDO or putrescineimpurities generated in diverging away from the pathway at any point.Isotopic enrichment can be achieved for any target atom including, forexample, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus,chloride or other halogens.

In some embodiments, the uptake sources can be selected to alter thecarbon-12, carbon-13, and carbon-14 ratios. In some embodiments, theuptake sources can be selected to alter the oxygen-16, oxygen-17, andoxygen-18 ratios. In some embodiments, the uptake sources can beselected to alter the hydrogen, deuterium, and tritium ratios. In someembodiments, the uptake sources can be selected to alter the nitrogen-14and nitrogen-15 ratios. In some embodiments, the uptake sources can beselected to alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35ratios. In some embodiments, the uptake sources can be selected to alterthe phosphorus-31, phosphorus-32, and phosphorus-33 ratios. In someembodiments, the uptake sources can be selected to alter thechlorine-35, chlorine-36, and chlorine-37 ratios.

In some embodiments, the isotopic ratio of a target atom can be variedto a desired ratio by selecting one or more uptake sources. An uptakesource can be derived from a natural source, as found in nature, or froma man-made source, and one skilled in the art can select a naturalsource, a man-made source, or a combination thereof, to achieve adesired isotopic ratio of a target atom. An example of a man-made uptakesource includes, for example, an uptake source that is at leastpartially derived from a chemical synthetic reaction. Such isotopicallyenriched uptake sources can be purchased commercially or prepared in thelaboratory and/or optionally mixed with a natural source of the uptakesource to achieve a desired isotopic ratio. In some embodiments, atarget atom isotopic ratio of an uptake source can be achieved byselecting a desired origin of the uptake source as found in nature. Forexample, as discussed herein, a natural source can be a biobased derivedfrom or synthesized by a biological organism or a source such aspetroleum-based products or the atmosphere. In some such embodiments, asource of carbon, for example, can be selected from a fossilfuel-derived carbon source, which can be relatively depleted ofcarbon-14, or an environmental or atmospheric carbon source, such asCO₂, which can possess a larger amount of carbon-14 than itspetroleum-derived counterpart.

The unstable carbon isotope carbon-14 or radiocarbon makes up forroughly 1 in 10¹² carbon atoms in the earth's atmosphere and has ahalf-life of about 5700 years. The stock of carbon is replenished in theupper atmosphere by a nuclear reaction involving cosmic rays andordinary nitrogen (¹⁴N). Fossil fuels contain no carbon-14, as itdecayed long ago. Burning of fossil fuels lowers the atmosphericcarbon-14 fraction, the so-called “Suess effect”.

Methods of determining the isotopic ratios of atoms in a compound arewell known to those skilled in the art. Isotopic enrichment is readilyassessed by mass spectrometry using techniques known in the art such asaccelerated mass spectrometry (AMS), Stable Isotope Ratio MassSpectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation byNuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques canbe integrated with separation techniques such as liquid chromatography(LC), high performance liquid chromatography (HPLC) and/or gaschromatography, and the like.

In the case of carbon, ASTM D6866 was developed in the United States asa standardized analytical method for determining the biobased content ofsolid, liquid, and gaseous samples using radiocarbon dating by theAmerican Society for Testing and Materials (ASTM) International. Thestandard is based on the use of radiocarbon dating for the determinationof a product's biobased content. ASTM D6866 was first published in 2004,and the current active version of the standard is ASTM D6866-11(effective Apr. 1, 2011). Radiocarbon dating techniques are well knownto those skilled in the art, including those described herein.

The biobased content of a compound is estimated by the ratio ofcarbon-14 (¹⁴C) to carbon-12 (¹²C). Specifically, the Fraction Modern(Fm) is computed from the expression: Fm=(S−B)/(M−B), where B, S and Mrepresent the ¹⁴C/¹²C ratios of the blank, the sample and the modernreference, respectively. Fraction Modern is a measurement of thedeviation of the ¹⁴C/¹²C ratio of a sample from “Modern.” Modern isdefined as 95% of the radiocarbon concentration (in AD 1950) of NationalBureau of Standards (NBS) Oxalic Acid I (i.e., standard referencematerials (SRM) 4990b) normalized to δ¹³C_(VPDB)=−19 per mil. Olsson,The use of Oxalic acid as a Standard. in, Radiocarbon Variations andAbsolute Chronology, Nobel Symposium, 12th Proc., John Wiley & Sons, NewYork (1970). Mass spectrometry results, for example, measured by ASM,are calculated using the internationally agreed upon definition of 0.95times the specific activity of NBS Oxalic Acid I (SRM 4990b) normalizedto δ¹³C_(VPDB)=−19 per mil. This is equivalent to an absolute (AD 1950)¹⁴C/¹²C ratio of 1.176±0.010×10⁻¹² (Karlen et al., Arkiv Geofysik,4:465-471 (1968)). The standard calculations take into account thedifferential uptake of one istope with respect to another, for example,the preferential uptake in biological systems of C¹² over C¹³ over C¹⁴,and these corrections are reflected as a Fm corrected for δ¹³.

An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of1955 sugar beet. Although there were 1000 lbs made, this oxalic acidstandard is no longer commercially available. The Oxalic Acid IIstandard (HOx 2; N.I.S.T designation SRM 4990 C) was made from a crop of1977 French beet molasses. In the early 1980's, a group of 12laboratories measured the ratios of the two standards. The ratio of theactivity of Oxalic acid II to 1 is 1.2933±0.001 (the weighted mean). Theisotopic ratio of HOx II is −17.8 per mille. ASTM D6866-11 suggests useof the available Oxalic Acid II standard SRM 4990 C (Hox2) for themodern standard (see discussion of original vs. currently availableoxalic acid standards in Mann, Radiocarbon, 25(2):519-527 (1983)). AFm=0% represents the entire lack of carbon-14 atoms in a material, thusindicating a fossil (for example, petroleum based) carbon source. AFm=100%, after correction for the post-1950 injection of carbon-14 intothe atmosphere from nuclear bomb testing, indicates an entirely moderncarbon source. As described herein, such a “modern” source includesbiobased sources.

As described in ASTM D6866, the percent modern carbon (pMC) can begreater than 100% because of the continuing but diminishing effects ofthe 1950s nuclear testing programs, which resulted in a considerableenrichment of carbon-14 in the atmosphere as described in ASTM D6866-11.Because all sample carbon-14 activities are referenced to a “pre-bomb”standard, and because nearly all new biobased products are produced in apost-bomb environment, all pMC values (after correction for isotopicfraction) must be multiplied by 0.95 (as of 2010) to better reflect thetrue biobased content of the sample. A biobased content that is greaterthan 103% suggests that either an analytical error has occurred, or thatthe source of biobased carbon is more than several years old.

ASTM D6866 quantifies the biobased content relative to the material'stotal organic content and does not consider the inorganic carbon andother non-carbon containing substances present. For example, a productthat is 50% starch-based material and 50% water would be considered tohave a Biobased Content=100% (50% organic content that is 100% biobased)based on ASTM D6866. In another example, a product that is 50%starch-based material, 25% petroleum-based, and 25% water would have aBiobased Content=66.7% (75% organic content but only 50% of the productis biobased). In another example, a product that is 50% organic carbonand is a petroleum-based product would be considered to have a BiobasedContent=0% (50% organic carbon but from fossil sources). Thus, based onthe well known methods and known standards for determining the biobasedcontent of a compound or material, one skilled in the art can readilydetermine the biobased content and/or prepared downstream products thatutilize of the invention having a desired biobased content.

Applications of carbon-14 dating techniques to quantify bio-basedcontent of materials are known in the art (Currie et al., NuclearInstruments and Methods in Physics Research B, 172:281-287 (2000)). Forexample, carbon-14 dating has been used to quantify bio-based content interephthalate-containing materials (Colonna et al., Green Chemistry,13:2543-2548 (2011)). Notably, polypropylene terephthalate (PPT)polymers derived from renewable 1,3-propanediol and petroleum-derivedterephthalic acid resulted in Fm values near 30% (i.e., since 3/11 ofthe polymeric carbon derives from renewable 1,3-propanediol and 8/11from the fossil end member terephthalic acid) (Currie et al., supra,2000). In contrast, polybutylene terephthalate polymer derived from bothrenewable 1,4-butanediol and renewable terephthalic acid resulted inbio-based content exceeding 90% (Colonna et al., supra, 2011).

Accordingly, in some embodiments, the present invention provides 4-HB,4-HBal, 4-HBCoA, BDO or putrescine or a 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine intermediate that has a carbon-12, carbon-13, and carbon-14ratio that reflects an atmospheric carbon uptake source. For example, insome aspects the 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine or a 4-HB,4-HBal, 4-HBCoA, BDO or putrescine intermediate can have an Fm value ofat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98% or as much as 100%.In some such embodiments, the uptake source is CO₂. In some embodiments,the present invention provides 4-HB, 4-HBal, 4-HBCoA, BDO or putrescineor a 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine intermediate that has acarbon-12, carbon-13, and carbon-14 ratio that reflects petroleum-basedcarbon uptake source. In this aspect, the 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine or a 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine intermediatecan have an Fm value of less than 95%, less than 90%, less than 85%,less than 80%, less than 75%, less than 70%, less than 65%, less than60%, less than 55%, less than 50%, less than 45%, less than 40%, lessthan 35%, less than 30%, less than 25%, less than 20%, less than 15%,less than 10%, less than 5%, less than 2% or less than 1%. In someembodiments, the present invention provides 4-HB, 4-HBal, 4-HBCoA, BDOor putrescine or a 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine intermediatethat has a carbon-12, carbon-13, and carbon-14 ratio that is obtained bya combination of an atmospheric carbon uptake source with apetroleum-based uptake source. Using such a combination of uptakesources is one way by which the carbon-12, carbon-13, and carbon-14ratio can be varied, and the respective ratios would reflect theproportions of the uptake sources.

Further, the present invention relates to the biologically produced4-HB, 4-HBal, 4-HBCoA, BDO or putrescine or 4-HB, 4-HBal, 4-HBCoA, BDOor putrescine intermediate as disclosed herein, and to the productsderived therefrom, wherein the 4-HB, 4-HBal, 4-HBCoA, BDO or putrescineor a 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine intermediate has acarbon-12, carbon-13, and carbon-14 isotope ratio of about the samevalue as the CO₂ that occurs in the environment. For example, in someaspects the invention provides: bioderived 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine or a bioderived 4-HB, 4-HBal, 4-HBCoA, BDO or putrescineintermediate having a carbon-12 versus carbon-13 versus carbon-14isotope ratio of about the same value as the CO₂ that occurs in theenvironment, or any of the other ratios disclosed herein. It isunderstood, as disclosed herein, that a product can have a carbon-12versus carbon-13 versus carbon-14 isotope ratio of about the same valueas the CO₂ that occurs in the environment, or any of the ratiosdisclosed herein, wherein the product is generated from bioderived 4-HB,4-HBal, 4-HBCoA, BDO or putrescine or a bioderived 4-HB, 4-HBal,4-HBCoA, BDO or putrescine intermediate as disclosed herein, wherein thebioderived product is chemically modified to generate a final product.Methods of chemically modifying a bioderived product of 4-HB, 4-HBal,4-HBCoA, BDO or putrescine, or an intermediate thereof, to generate adesired product are well known to those skilled in the art, as describedherein. The invention further provides plastics, elastic fibers,polyurethanes, polyesters, including polyhydroxyalkanoates such aspoly-4-hydroxybutyrate (P4HB) or co-polymers thereof,poly(tetramethylene ether) glycol (PTMEG) (also referred to as PTMO,polytetramethylene oxide) and polyurethane-polyurea copolymers, referredto as spandex, elastane or Lycra™, nylons, and the like, having acarbon-12 versus carbon-13 versus carbon-14 isotope ratio of about thesame value as the CO₂ that occurs in the environment, wherein theplastics, elastic fibers, polyurethanes, polyesters, includingpolyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) orco-polymers thereof, poly(tetramethylene ether) glycol (PTMEG) (alsoreferred to as PTMO, polytetramethylene oxide) and polyurethane-polyureacopolymers, referred to as spandex, elastane or Lycra™, and nylons aregenerated directly from or in combination with bioderived 4-HB, 4-HBal,4-HBCoA, BDO or putrescine or a bioderived 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine intermediate as disclosed herein.

The products 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine are chemicalscommonly used in many commercial and industrial applications.Non-limiting examples of such applications include production ofplastics, elastic fibers, polyurethanes, polyesters, includingpolyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) orco-polymers thereof, poly(tetramethylene ether) glycol (PTMEG) (alsoreferred to as PTMO, polytetramethylene oxide) and polyurethane-polyureacopolymers, referred to as spandex, elastane or Lycra™, and nylons.Moreover, 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine are also used as araw material in the production of a wide range of products includingplastics, elastic fibers, polyurethanes, polyesters, includingpolyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) orco-polymers thereof, poly(tetramethylene ether) glycol (PTMEG) (alsoreferred to as PTMO, polytetramethylene oxide) and polyurethane-polyureacopolymers, referred to as spandex, elastane or Lycra™, nylons.Accordingly, in some embodiments, the invention provides biobasedplastics, elastic fibers, polyurethanes, polyesters, includingpolyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) orco-polymers thereof, poly(tetramethylene ether) glycol (PTMEG) (alsoreferred to as PTMO, polytetramethylene oxide) and polyurethane-polyureacopolymers, referred to as spandex, elastane or Lycra™, nylonscomprising one or more bioderived 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine or bioderived 4-HB, 4-HBal, 4-HBCoA, BDO or putrescineintermediate produced by a non-naturally occurring microorganism of theinvention or produced using a method disclosed herein.

As used herein, the term “bioderived” means derived from or synthesizedby a biological organism and can be considered a renewable resourcesince it can be generated by a biological organism. Such a biologicalorganism, in particular the microbial organisms of the inventiondisclosed herein, can utilize feedstock or biomass, such as, sugars orcarbohydrates obtained from an agricultural, plant, bacterial, or animalsource. Alternatively, the biological organism can utilize atmosphericcarbon. As used herein, the term “biobased” means a product as describedabove that is composed, in whole or in part, of a bioderived compound ofthe invention. A biobased or bioderived product is in contrast to apetroleum derived product, wherein such a product is derived from orsynthesized from petroleum or a petrochemical feedstock.

In some embodiments, the invention provides plastics, elastic fibers,polyurethanes, polyesters, including polyhydroxyalkanoates such aspoly-4-hydroxybutyrate (P4HB) or co-polymers thereof,poly(tetramethylene ether) glycol (PTMEG) (also referred to as PTMO,polytetramethylene oxide) and polyurethane-polyurea copolymers, referredto as spandex, elastane or Lycra™, nylons comprising bioderived 4-HB,4-HBal, 4-HBCoA, BDO or putrescine or bioderived 4-HB, 4-HBal, 4-HBCoA,BDO or putrescine intermediate, wherein the bioderived 4-HB, 4-HBal,4-HBCoA, BDO or putrescine or bioderived 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine intermediate includes all or part of the 4-HB, 4-HBal,4-HBCoA, BDO or putrescine or 4-HB, 4-HBal, 4-HBCoA, BDO or putrescineintermediate used in the production of plastics, elastic fibers,polyurethanes, polyesters, including polyhydroxyalkanoates such aspoly-4-hydroxybutyrate (P4HB) or co-polymers thereof,poly(tetramethylene ether) glycol (PTMEG) (also referred to as PTMO,polytetramethylene oxide) and polyurethane-polyurea copolymers, referredto as spandex, elastane or Lycra™, and nylons. Thus, in some aspects,the invention provides a biobased plastics, elastic fibers,polyurethanes, polyesters, including polyhydroxyalkanoates such aspoly-4-hydroxybutyrate (P4HB) or co-polymers thereof,poly(tetramethylene ether) glycol (PTMEG) (also referred to as PTMO,polytetramethylene oxide) and polyurethane-polyurea copolymers, referredto as spandex, elastane or Lycra™, and nylons comprising at least 2%, atleast 3%, at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98% or 100% bioderived 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine orbioderived 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine intermediate asdisclosed herein. Additionally, in some aspects, the invention providesa biobased plastics, elastic fibers, polyurethanes, polyesters,including polyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) orco-polymers thereof, poly(tetramethylene ether) glycol (PTMEG) (alsoreferred to as PTMO, polytetramethylene oxide) and polyurethane-polyureacopolymers, referred to as spandex, elastane or Lycra™, and nylonswherein the 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine or 4-HB, 4-HBal,4-HBCoA, BDO or putrescine intermediate used in its production is acombination of bioderived and petroleum derived 4-HB, 4-HBal, 4-HBCoA,BDO or putrescine or 4-HB, 4-HBal, 4-HBCoA, BDO or putrescineintermediate. For example, a biobased plastics, elastic fibers,polyurethanes, polyesters, including polyhydroxyalkanoates such aspoly-4-hydroxybutyrate (P4HB) or co-polymers thereof,poly(tetramethylene ether) glycol (PTMEG) (also referred to as PTMO,polytetramethylene oxide) and polyurethane-polyurea copolymers, referredto as spandex, elastane or Lycra™, and nylons can be produced using 50%bioderived the 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine and 50%petroleum derived 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine or otherdesired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%,100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleumderived precursors, so long as at least a portion of the productcomprises a bioderived product produced by the microbial organismsdisclosed herein. It is understood that methods for producing plastics,elastic fibers, polyurethanes, polyesters, includingpolyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) orco-polymers thereof, poly(tetramethylene ether) glycol (PTMEG) (alsoreferred to as PTMO, polytetramethylene oxide) and polyurethane-polyureacopolymers, referred to as spandex, elastane or Lycra™, and nylons usingthe bioderived 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine or bioderived4-HB, 4-HBal, 4-HBCoA, BDO or putrescine intermediate of the inventionare well known in the art.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described herein, particularly useful yields of thebiosynthetic products of the invention can be obtained under anaerobicor substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achievingbiosynthesis of 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine includesanaerobic culture or fermentation conditions. In certain embodiments,the non-naturally occurring microbial organisms of the invention can besustained, cultured or fermented under anaerobic or substantiallyanaerobic conditions. Briefly, anaerobic conditions refers to anenvironment devoid of oxygen. Substantially anaerobic conditionsinclude, for example, a culture, batch fermentation or continuousfermentation such that the dissolved oxygen concentration in the mediumremains between 0 and 10% of saturation. Substantially anaerobicconditions also includes growing or resting cells in liquid medium or onsolid agar inside a sealed chamber maintained with an atmosphere of lessthan 1% oxygen. The percent of oxygen can be maintained by, for example,sparging the culture with an N₂/CO₂ mixture or other suitable non-oxygengas or gases.

The invention also provides a non-naturally occurring microbialbiocatalyst including a microbial organism having 4-hydroxybutanoic acid(4-HB) and 1,4-butanediol (BDO) biosynthetic pathways that include atleast one exogenous nucleic acid encoding 4-hydroxybutanoatedehydrogenase, CoA-independent succinic semialdehyde dehydrogenase,succinyl-CoA synthetase, CoA-dependent succinic semialdehydedehydrogenase, 4-hydroxybutyrate:CoA transferase, glutamate: succinicsemialdehyde transaminase, glutamate decarboxylase, CoA-independentaldehyde dehydrogenase, CoA-dependent aldehyde dehydrogenase or alcoholdehydrogenase, wherein the exogenous nucleic acid is expressed insufficient amounts to produce 1,4-butanediol (BDO).4-Hydroxybutyrate:CoA transferase also is known as 4-hydroxybutyrylCoA:acetyl-CoA transferase. Additional 4-HB or BDO pathway enzymes arealso disclosed herein (see Examples and FIGS. 8-13).

The invention further provides non-naturally occurring microbialbiocatalyst including a microbial organism having 4-hydroxybutanoic acid(4-HB) and 1,4-butanediol (BDO) biosynthetic pathways, the pathwaysinclude at least one exogenous nucleic acid encoding 4-hydroxybutanoatedehydrogenase, succinyl-CoA synthetase, CoA-dependent succinicsemialdehyde dehydrogenase, 4-hydroxybutyrate:CoA transferase,4-butyrate kinase, phosphotransbutyrylase, α-ketoglutaratedecarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase or analdehyde/alcohol dehydrogenase, wherein the exogenous nucleic acid isexpressed in sufficient amounts to produce 1,4-butanediol (BDO).

Non-naturally occurring microbial organisms also can be generated whichbiosynthesize BDO. As with the 4-HB producing microbial organisms of theinvention, the BDO producing microbial organisms also can produceintracellularly or secret the BDO into the culture medium. Following theteachings and guidance provided previously for the construction ofmicrobial organisms that synthesize 4-HB, additional BDO pathways can beincorporated into the 4-HB producing microbial organisms to generateorganisms that also synthesize BDO and other BDO family compounds. Thechemical synthesis of BDO and its downstream products are known. Thenon-naturally occurring microbial organisms of the invention capable ofBDO biosynthesis circumvent these chemical synthesis using 4-HB as anentry point as illustrated in FIG. 1. As described further below, the4-HB producers also can be used to chemically convert 4-HB to GBL andthen to BDO or THF, for example. Alternatively, the 4-HB producers canbe further modified to include biosynthetic capabilities for conversionof 4-HB and/or GBL to BDO.

The additional BDO pathways to introduce into 4-HB producers include,for example, the exogenous expression in a host deficient background orthe overexpression of one or more of the enzymes exemplified in FIG. 1as steps 9-13. One such pathway includes, for example, the enzymeactivities necessary to carryout the reactions shown as steps 9, 12 and13 in FIG. 1, where the aldehyde and alcohol dehydrogenases can beseparate enzymes or a multifunctional enzyme having both aldehyde andalcohol dehydrogenase activity. Another such pathway includes, forexample, the enzyme activities necessary to carry out the reactionsshown as steps 10, 11, 12 and 13 in FIG. 1, also where the aldehyde andalcohol dehydrogenases can be separate enzymes or a multifunctionalenzyme having both aldehyde and alcohol dehydrogenase activity.Accordingly, the additional BDO pathways to introduce into 4-HBproducers include, for example, the exogenous expression in a hostdeficient background or the overexpression of one or more of a4-hydroxybutyrate:CoA transferase, butyrate kinase,phosphotransbutyrylase, CoA-independent aldehyde dehydrogenase,CoA-dependent aldehyde dehydrogenase or an alcohol dehydrogenase. In theabsence of endogenous acyl-CoA synthetase capable of modifying 4-HB, thenon-naturally occurring BDO producing microbial organisms can furtherinclude an exogenous acyl-CoA synthetase selective for 4-HB, or thecombination of multiple enzymes that have as a net reaction conversionof 4-HB into 4-HB-CoA. As exemplified further below in the Examples,butyrate kinase and phosphotransbutyrylase exhibit BDO pathway activityand catalyze the conversions illustrated in FIG. 1 with a 4-HBsubstrate. Therefore, these enzymes also can be referred to herein as4-hydroxybutyrate kinase and phosphotranshydroxybutyrylase respectively.

Exemplary alcohol and aldehyde dehydrogenases that can be used for thesein vivo conversions from 4-HB to BDO are listed below in Table 1.

TABLE 1 Alcohol and Aldehyde Dehydrogenases for Conversion of 4-HB toBDO. ALCOHOL DEHYDROGENASES ec: 1.1.1.1 alcohol dehydrogenase ec:1.1.1.2 alcohol dehydrogenase (NADP+) ec: 1.1.1.4 (R,R)-butanedioldehydrogenase ec: 1.1.1.5 acetoin dehydrogenase ec: 1.1.1.6 glyceroldehydrogenase ec: 1.1.1.7 propanediol-phosphate dehydrogenase ec:1.1.1.8 glycerol-3-phosphate dehydrogenase (NAD+) ec: 1.1.1.11D-arabinitol 4-dehydrogenase ec: 1.1.1.12 L-arabinitol 4-dehydrogenaseec: 1.1.1.13 L-arabinitol 2-dehydrogenase ec: 1.1.1.14 L-iditol2-dehydrogenase ec: 1.1.1.15 D-iditol 2-dehydrogenase ec: 1.1.1.16galactitol 2-dehydrogenase ec: 1.1.1.17 mannitol-1-phosphate5-dehydrogenase ec: 1.1.1.18 inositol 2-dehydrogenase ec: 1.1.1.21aldehyde reductase ec: 1.1.1.23 histidinol dehydrogenase ec: 1.1.1.26glyoxylate reductase ec: 1.1.1.27 L-lactate dehydrogenase ec: 1.1.1.28D-lactate dehydrogenase ec: 1.1.1.29 glycerate dehydrogenase ec:1.1.1.30 3-hydroxybutyrate dehydrogenase ec: 1.1.1.313-hydroxyisobutyrate dehydrogenase ec: 1.1.1.35 3-hydroxyacyl-CoAdehydrogenase ec: 1.1.1.36 acetoacetyl-CoA reductase ec: 1.1.1.37 malatedehydrogenase ec: 1.1.1.38 malate dehydrogenase (oxaloacetate-decarboxylating) ec: 1.1.1.39 malate dehydrogenase (decarboxylating) ec:1.1.1.40 malate dehydrogenase (oxaloacetate- decarboxylating) (NADP+)ec: 1.1.1.41 isocitrate dehydrogenase (NAD+) ec: 1.1.1.42 isocitratedehydrogenase (NADP+) ec: 1.1.1.54 allyl-alcohol dehydrogenase ec:1.1.1.55 lactaldehyde reductase (NADPH) ec: 1.1.1.56 ribitol2-dehydrogenase ec: 1.1.1.59 3-hydroxypropionate dehydrogenase ec:1.1.1.60 2-hydroxy-3-oxopropionate reductase ec: 1.1.1.614-hydroxybutyrate dehydrogenase ec: 1.1.1.66 omega-hydroxydecanoatedehydrogenase ec: 1.1.1.67 mannitol 2-dehydrogenase ec: 1.1.1.71 alcoholdehydrogenase [NAD(P)+] ec: 1.1.1.72 glycerol dehydrogenase (NADP+) ec:1.1.1.73 octanol dehydrogenase ec: 1.1.1.75 (R)-aminopropanoldehydrogenase ec: 1.1.1.76 (S,S)-butanediol dehydrogenase ec: 1.1.1.77lactaldehyde reductase ec: 1.1.1.78 methylglyoxal reductase (NADH-dependent) ec: 1.1.1.79 glyoxylate reductase (NADP+) ec: 1.1.1.80isopropanol dehydrogenase (NADP+) ec: 1.1.1.81 hydroxypyruvate reductaseec: 1.1.1.82 malate dehydrogenase (NADP+) ec: 1.1.1.83 D-malatedehydrogenase (decarboxylating) ec: 1.1.1.84 dimethylmalatedehydrogenase ec: 1.1.1.85 3-isopropylmalate dehydrogenase ec: 1.1.1.86ketol-acid reductoisomerase ec: 1.1.1.87 homoisocitrate dehydrogenaseec: 1.1.1.88 hydroxymethylglutaryl-CoA reductase ec: 1.1.1.90aryl-alcohol dehydrogenase ec: 1.1.1.91 aryl-alcohol dehydrogenase(NADP+) ec: 1.1.1.92 oxaloglycolate reductase (decarboxylating) ec:1.1.1.94 glycerol-3-phosphate dehydrogenase [NAD(P)+] ec: 1.1.1.95phosphoglycerate dehydrogenase ec: 1.1.1.97 3-hydroxybenzyl-alcoholdehydrogenase ec: 1.1.1.101 acylglycerone-phosphate reductase ec:1.1.1.103 L-threonine 3-dehydrogenase ec: 1.1.1.104 4-oxoprolinereductase ec: 1.1.1.105 retinol dehydrogenase ec: 1.1.1.110indolelactate dehydrogenase ec: 1.1.1.112 indanol dehydrogenase ec:1.1.1.113 L-xylose 1-dehydrogenase ec: 1.1.1.129 L-threonate3-dehydrogenase ec: 1.1.1.137 ribitol-5-phosphate 2-dehydrogenase ec:1.1.1.138 mannitol 2-dehydrogenase (NADP+) ec: 1.1.1.140sorbitol-6-phosphate 2-dehydrogenase ec: 1.1.1.142 D-pinitoldehydrogenase ec: 1.1.1.143 sequoyitol dehydrogenase ec: 1.1.1.144perillyl-alcohol dehydrogenase ec: 1.1.1.156 glycerol 2-dehydrogenase(NADP+) ec: 1.1.1.157 3-hydroxybutyryl-CoA dehydrogenase ec: 1.1.1.163cyclopentanol dehydrogenase ec: 1.1.1.164 hexadecanol dehydrogenase ec:1.1.1.165 2-alkyn-1-ol dehydrogenase ec: 1.1.1.166hydroxycyclohexanecarboxylate dehydrogenase ec: 1.1.1.167hydroxymalonate dehydrogenase ec: 1.1.1.174 cyclohexane-1,2-dioldehydrogenase ec: 1.1.1.177 glycerol-3-phosphate 1-dehydrogenase (NADP+)ec: 1.1.1.178 3-hydroxy-2-methylbutyryl-CoA dehydrogenase ec: 1.1.1.185L-glycol dehydrogenase ec: 1.1.1.190 indole-3-acetaldehyde reductase(NADH) ec: 1.1.1.191 indole-3-acetaldehyde reductase (NADPH) ec:1.1.1.192 long-chain-alcohol dehydrogenase ec: 1.1.1.194coniferyl-alcohol dehydrogenase ec: 1.1.1.195 cinnamyl-alcoholdehydrogenase ec: 1.1.1.198 (+)-borneol dehydrogenase ec: 1.1.1.2021,3-propanediol dehydrogenase ec: 1.1.1.207 (−)-menthol dehydrogenaseec: 1.1.1.208 (+)-neomenthol dehydrogenase ec: 1.1.1.216 farnesoldehydrogenase ec: 1.1.1.217 benzyl-2-methyl-hydroxybutyratedehydrogenase ec: 1.1.1.222 (R)-4-hydroxyphenyllactate dehydrogenase ec:1.1.1.223 isopiperitenol dehydrogenase ec: 1.1.1.2264-hydroxycyclohexanecarboxylate dehydrogenase ec: 1.1.1.229 diethyl2-methyl-3-oxosuccinate reductase ec: 1.1.1.237 hydroxyphenylpyruvatereductase ec: 1.1.1.244 methanol dehydrogenase ec: 1.1.1.245cyclohexanol dehydrogenase ec: 1.1.1.250 D-arabinitol 2-dehydrogenaseec: 1.1.1.251 galactitol 1-phosphate 5-dehydrogenase ec: 1.1.1.255mannitol dehydrogenase ec: 1.1.1.256 fluoren-9-ol dehydrogenase ec:1.1.1.257 4-(hydroxymethyl)benzenesulfonate dehydrogenase ec: 1.1.1.2586-hydroxyhexanoate dehydrogenase ec: 1.1.1.259 3-hydroxypimeloyl-CoAdehydrogenase ec: 1.1.1.261 glycerol-1-phosphate dehydrogenase [NAD(P)+]ec: 1.1.1.265 3-methylbutanal reductase ec: 1.1.1.283 methylglyoxalreductase (NADPH- dependent) ec: 1.1.1.286 isocitrate-homoisocitratedehydrogenase ec: 1.1.1.287 D-arabinitol dehydrogenase (NADP+) butanoldehydrogenase ALDEHYDE DEHYDROGENASES ec: 1.2.1.2 formate dehydrogenaseec: 1.2.1.3 aldehyde dehydrogenase (NAD+) ec: 1.2.1.4 aldehydedehydrogenase (NADP+) ec: 1.2.1.5 aldehyde dehydrogenase [NAD(P)+] ec:1.2.1.7 benzaldehyde dehydrogenase (NADP+) ec: 1.2.1.8 betaine-aldehydedehydrogenase ec: 1.2.1.9 glyceraldehyde-3-phosphate dehydrogenase(NADP+) ec: 1.2.1.10 acetaldehyde dehydrogenase (acetylating) ec:1.2.1.11 aspartate-semialdehyde dehydrogenase ec: 1.2.1.12glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) ec: 1.2.1.13glyceraldehyde-3-phosphate dehydrogenase (NADP+) (phosphorylating) ec:1.2.1.15 malonate-semialdehyde dehydrogenase ec: 1.2.1.16succinate-semialdehyde dehydrogenase [NAD(P)+] ec: 1.2.1.17 glyoxylatedehydrogenase (acylating) ec: 1.2.1.18 malonate-semialdehydedehydrogenase (acetylating) ec: 1.2.1.19 aminobutyraldehydedehydrogenase ec: 1.2.1.20 glutarate-semialdehyde dehydrogenase ec:1.2.1.21 glycolaldehyde dehydrogenase ec: 1.2.1.22 lactaldehydedehydrogenase ec: 1.2.1.23 2-oxoaldehyde dehydrogenase (NAD+) ec:1.2.1.24 succinate-semialdehyde dehydrogenase ec: 1.2.1.252-oxoisovalerate dehydrogenase (acylating) ec: 1.2.1.262,5-dioxovalerate dehydrogenase ec: 1.2.1.27 methylmalonate-semialdehydedehydrogenase (acylating) ec: 1.2.1.28 benzaldehyde dehydrogenase (NAD+)ec: 1.2.1.29 aryl-aldehyde dehydrogenase ec: 1.2.1.30 aryl-aldehydedehydrogenase (NADP+) ec: 1.2.1.31 L-aminoadipate-semialdehydedehydrogenase ec: 1.2.1.32 aminomuconate-semialdehyde dehydrogenase ec:1.2.1.36 retinal dehydrogenase ec: 1.2.1.39 phenylacetaldehydedehydrogenase ec: 1.2.1.41 glutamate-5-semialdehyde dehydrogenase ec:1.2.1.42 hexadecanal dehydrogenase (acylating) ec: 1.2.1.43 formatedehydrogenase (NADP+) ec: 1.2.1.45 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase ec: 1.2.1.46 formaldehyde dehydrogenase ec:1.2.1.47 4-trimethylammoniobutyraldehyde dehydrogenase ec: 1.2.1.48long-chain-aldehyde dehydrogenase ec: 1.2.1.49 2-oxoaldehydedehydrogenase (NADP+) ec: 1.2.1.51 pyruvate dehydrogenase (NADP+) ec:1.2.1.52 oxoglutarate dehydrogenase (NADP+) ec: 1.2.1.534-hydroxyphenylacetaldehyde dehydrogenase ec: 1.2.1.57 butanaldehydrogenase ec: 1.2.1.58 phenylglyoxylate dehydrogenase (acylating)ec: 1.2.1.59 glyceraldehyde-3-phosphate dehydrogenase (NAD(P)+)(phosphorylating) ec: 1.2.1.62 4-formylbenzenesulfonate dehydrogenaseec: 1.2.1.63 6-oxohexanoate dehydrogenase ec: 1.2.1.644-hydroxybenzaldehyde dehydrogenase ec: 1.2.1.65 salicylaldehydedehydrogenase ec: 1.2.1.66 mycothiol-dependent formaldehydedehydrogenase ec: 1.2.1.67 vanillin dehydrogenase ec: 1.2.1.68coniferyl-aldehyde dehydrogenase ec: 1.2.1.69 fluoroacetaldehydedehydrogenase ec: 1.2.1.71 succinylglutamate-semialdehyde dehydrogenas

Other exemplary enzymes and pathways are disclosed herein (seeExamples). Furthermore, it is understood that enzymes can be utilizedfor carry out reactions for which the substrate is not the naturalsubstrate. While the activity for the non-natural substrate may be lowerthan the natural substrate, it is understood that such enzymes can beutilized, either as naturally occurring or modified using the directedevolution or adaptive evolution, as disclosed herein (see alsoExamples).

BDO production through any of the pathways disclosed herein are based,in part, on the identification of the appropriate enzymes for conversionof precursors to BDO. A number of specific enzymes for several of thereaction steps have been identified. For those transformations whereenzymes specific to the reaction precursors have not been identified,enzyme candidates have been identified that are best suited forcatalyzing the reaction steps. Enzymes have been shown to operate on abroad range of substrates, as discussed below. In addition, advances inthe field of protein engineering also make it feasible to alter enzymesto act efficiently on substrates, even if not a natural substrate.Described below are several examples of broad-specificity enzymes fromdiverse classes suitable for a BDO pathway as well as methods that havebeen used for evolving enzymes to act on non-natural substrates.

A key class of enzymes in BDO pathways is the oxidoreductases thatinterconvert ketones or aldehydes to alcohols (1.1.1). Numerousexemplary enzymes in this class can operate on a wide range ofsubstrates. An alcohol dehydrogenase (1.1.1.1) purified from the soilbacterium Brevibacterium sp KU 1309 (Hirano et al., J. Biosc. Bioeng.100:318-322 (2005)) was shown to operate on a plethora of aliphatic aswell as aromatic alcohols with high activities. Table 2 shows theactivity of the enzyme and its K_(m) on different alcohols. The enzymeis reversible and has very high activity on several aldehydes also(Table 3).

TABLE 2 Relative activities of an alcohol dehydrogenase fromBrevibacterium sp KU to oxidize various alcohols. Relative ActivityK_(m) Substrate (0%) (mM) 2-Phenylethanol 100* 0.025(S)-2-Phenylpropanol 156  0.157 (R)-2-Phenylpropanol 63 0.020 Bynzylalcohol 199  0.012 3-Phenylpropanol 135  0.033 Ethanol 76 1-Butanol 111 1-Octanol 101  1-Dodecanol 68 1-Phenylethanol 46 2-Propanol 54 *Theactivity of 2-phenylethanol, corresponding to 19.2 U/mg, was taken as100%.

TABLE 3 Relative activities of an alcohol dehydrogenase fromBrevibacterium sp KU 1309 to reduce various carbonyl compounds. RelativeActivity K_(m) Substrate (%) (mM) Phenylacetaldehyde 100 0.2612-Phenylpropionaldehyde 188 0.864 1-Octylaldehyde 87 Acetophenone 0

Lactate dehydrogenase (1.1.1.27) from Ralstonia eutropha is anotherenzyme that has been demonstrated to have high activities on several2-oxoacids such as 2-oxobutyrate, 2-oxopentanoate and 2-oxoglutarate (aC5 compound analogous to 2-oxoadipate) (Steinbuchel and Schlegel, Eur.J. Biochem. 130:329-334 (1983)). Column 2 in Table 4 demonstrates theactivities of ldhA from R. eutropha (formerly A. eutrophus) on differentsubstrates (Steinbuchel and Schlegel, supra, 1983).

TABLE 4 The in vitro activity ofR. eutropha ldhA (Steinbuchel andSchlegel, supra, 1983) on different substrates and compared with that onpyruvate. Activity (%) of L(+)-lactate L(+)-lactate D(−)-lactatedehydrogenase dehydrogenase dehydrogenase from from from Substrate A.eutrophus rabbit muscle L. leichmanii Glyoxylate 8.7 23.9 5.0 Pyruvate100.0 100.0 100.0 2-Oxobutyrate 107.0 18.6 1.1 2-Oxovalerate 125.0 0.70.0 3-Methyl-2- 28.5 0.0 0.0 oxobutyrate 3-Methyl-2- 5.3 0.0 0.0oxovalerate 4-Methyl-2- 39.0 1.4 1.1 oxopentanoate Oxaloacetate 0.0 33.123.1 2-Oxoglutarate 79.6 0.0 0.0 3-Fluoropyruvate 33.6 74.3 40.0

Oxidoreductases that can convert 2-oxoacids to their acyl-CoAcounterparts (1.2.1) have been shown to accept multiple substrates aswell. For example, branched-chain 2-keto-acid dehydrogenase complex(BCKAD), also known as 2-oxoisovalerate dehydrogenase (1.2.1.25),participates in branched-chain amino acid degradation pathways,converting 2-keto acids derivatives of valine, leucine and isoleucine totheir acyl-CoA derivatives and CO₂. In some organisms including Rattusnorvegicus (Paxton et al., Biochem. J. 234:295-303 (1986)) andSaccharomyces cerevisiae (Sinclair et al., Biochem. Mol. Biol. Int.32:911-922 (1993), this complex has been shown to have a broad substraterange that includes linear oxo-acids such as 2-oxobutanoate andalpha-ketoglutarate, in addition to the branched-chain amino acidprecursors.

Members of yet another class of enzymes, namely aminotransferases(2.6.1), have been reported to act on multiple substrates. Aspartateaminotransferase (aspA 7) from Pyrococcus fursious has been identified,expressed in E. coli and the recombinant protein characterized todemonstrate that the enzyme has the highest activities towards aspartateand alpha-ketoglutarate but lower, yet significant activities towardsalanine, glutamate and the aromatic amino acids (Ward et al., Archaea133-141 (2002)). In another instance, an aminotransferase identifiedfrom Leishmania mexicana and expressed in E. coli (Vernal et al., FEMSMicrobiol. Lett. 229:217-222 (2003)) was reported to have a broadsubstrate specificity towards tyrosine (activity considered 100% ontyrosine), phenylalanine (90%), tryptophan (85%), aspartate (30%),leucine (25%) and methionine (25%), respectively (Vernal et al., Mol.Biochem. Parasitol 96:83-92 (1998)). Similar broad specificity has beenreported for a tyrosine aminotransferase from Trypanosoma cruzi, eventhough both of these enzymes have a sequence homology of only 6%. Thelatter enzyme can accept leucine, methionine as well as tyrosine,phenylalanine, tryptophan and alanine as efficient amino donors (Nowickiet al., Biochim. Biophys. Acta 1546: 268-281 (2001)).

CoA transferases (2.8.3) have been demonstrated to have the ability toact on more than one substrate. Specifically, a CoA transferase waspurified from Clostridium acetobutylicum and was reported to have thehighest activities on acetate, propionate, and butyrate. It also hadsignificant activities with valerate, isobutyrate, and crotonate(Wiesenborn et al., Appl. Environ. Microbiol. 55:323-329 (1989)). Inanother study, the E. coli enzyme acyl-CoA:acetate-CoA transferase, alsoknown as acetate-CoA transferase (EC 2.8.3.8), has been shown totransfer the CoA moiety to acetate from a variety of branched and linearacyl-CoA substrates, including isobutyrate (Matthies and Schink, App.Environm. Microbiol. 58:1435-1439 (1992)), valerate (Vanderwinkel etal., Biochem. Biophys. Res Commun. 33:902-908 (1968b)) and butanoate(Vanderwinkel et al., Biochem. Biophys. Res Commun. 33:902-908(1968a).

Other enzyme classes additionally support broad substrate specificityfor enzymes. Some isomerases (5.3.3) have also been proven to operate onmultiple substrates. For example, L-rhamnose isomerase from Pseudomonasstutzeri catalyzes the isomerization between various aldoalses andketoses (Yoshida et al., J. Mol. Biol. 365:1505-1516 (2007)). Theseinclude isomerization between L-rhamnose and L-rhamnulose, L-mannose andL-fructose, L-xylose and L-xylulose, D-ribose and D-ribulose, andD-allose and D-psicose.

In yet another class of enzymes, the phosphotransferases (2.7.1), thehomoserine kinase (2.7.1.39) from E. coli that converts L-homoserine toL-homoserine phosphate, was found to phosphorylate numerous homoserineanalogs. In these substrates, the carboxyl functional group at theR-position had been replaced by an ester or by a hydroxymethyl group(Huo and Viola, Biochemistry 35:16180-16185 (1996)). Table 5demonstrates the broad substrate specificity of this kinase.

TABLE 5 The substrate specificity of homoserine kinase. Substratek_(cat) % k_(cat) K_(m) (mM) k_(cat)/K_(m) L-homoserine 18.3 ± 0.1 1000.14 ± 0.04 184 ± 17  D-homoserine  8.3 ± 1.1 32 31.8 ± 7.2  0.26 ± 0.03L-aspartate β-  2.1 ± 0.1 8.2 0.28 ± 0.02 7.5 ± 0.3 semialdehydeL-2-amino-1,4-  2.0 ± 0.5 7.9 11.6 ± 6.5  0.17 ± 0.06 butanediolL-2-amino-5-  2.5 ± 0.4 9.9 1.1 ± 0.5 2.3 ± 0.3 hydroxyvalerateL-homoserine 14.7 ± 2.6 80 4.9 ± 2.0 3.0 ± 0.6 methyl ester L-homoserineethyl 13.6 ± 0.8 74 1.9 ± 0.5 7.2 ± 1.7 ester L-homoserine 13.6 ± 1.4 741.2 ± 0.5 11.3 ± 1.1  isopropyl ester L-homoserine n- 14.0 ± 0.4 76 3.5± 0.4 4.0 ± 1.2 propyl ester L-homoserine 16.4 ± 0.8 84 6.9 ± 1.1 2.4 ±0.3 isobutyl ester L-homserine n-butyl 29.1 ± 1.2 160 5.8 ± 0.8 5.0 ±0.5 ester

Another class of enzymes useful in BDO pathways is the acid-thiolligases (6.2.1). Like enzymes in other classes, certain enzymes in thisclass have been determined to have broad substrate specificity. Forexample, acyl CoA ligase from Pseudomonas putida has been demonstratedto work on several aliphatic substrates including acetic, propionic,butyric, valeric, hexanoic, heptanoic, and octanoic acids and onaromatic compounds such as phenylacetic and phenoxyacetic acids(Fernandez-Valverde et al., Appl. Environ. Microbiol. 59:1149-1154(1993)). A related enzyme, malonyl CoA synthetase (6.3.4.9) fromRhizobium trifolii could convert several diacids, namely, ethyl-,propyl-, allyl-, isopropyl-, dimethyl-, cyclopropyl-,cyclopropylmethylene-, cyclobutyl-, and benzyl-malonate into theircorresponding monothioesters (Pohl et al., J. Am. Chem. Soc.123:5822-5823 (2001)). Similarly, decarboxylases (4.1.1) have also beenfound with broad substrate ranges. Pyruvate decarboxylase (PDC), alsotermed keto-acid decarboxylase, is a key enzyme in alcoholicfermentation, catalyzing the decarboxylation of pyruvate toacetaldehyde. The enzyme isolated from Saccharomyces cerevisiae has abroad substrate range for aliphatic 2-keto acids including2-ketobutyrate, 2-ketovalerate, and 2-phenylpyruvate (L1 and Jordan,Biochemistry 38:10004-10012 (1999)). Similarly, benzoylformatedecarboxylase has a broad substrate range and has been the target ofenzyme engineering studies. The enzyme from Pseudomonas putida has beenextensively studied and crystal structures of this enzyme are available(Polovnikova et al., Biochemistry 42:1820-1830 (2003); Hasson et al.,Biochemistry 37:9918-9930 (1998)). Branched chain alpha-ketoaciddecarboxylase (BCKA) has been shown to act on a variety of compoundsvarying in chain length from 3 to 6 carbons (Oku and Kaneda, J. Biol.Chem. 263:18386-18396 (1998); Smit et al., Appl. Environ. Microbiol.71:303-311 (2005b)). The enzyme in Lactococcus lactis has beencharacterized on a variety of branched and linear substrates including2-oxobutanoate, 2-oxohexanoate, 2-oxopentanoate,3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate and isocaproate (Smitet al., Appl. Environ. Microbiol. 71:303-311 (2005a).

Interestingly, enzymes known to have one dominant activity have alsobeen reported to catalyze a very different function. For example, thecofactor-dependent phosphoglycerate mutase (5.4.2.1) from Bacillusstearothermophilus and Bacillus subtilis is known to function as aphosphatase as well (Rigden et al., Protein Sci. 10:1835-1846 (2001)).The enzyme from B. stearothermophilus is known to have activity onseveral substrates, including 3-phosphoglycerate,alpha-napthylphosphate, p-nitrophenylphosphate, AMP,fructose-6-phosphate, ribose-5-phosphate and CMP.

In contrast to these examples where the enzymes naturally have broadsubstrate specificities, numerous enzymes have been modified usingdirected evolution to broaden their specificity towards theirnon-natural substrates. Alternatively, the substrate preference of anenzyme has also been changed using directed evolution. Therefore, it isfeasible to engineer a given enzyme for efficient function on a natural,for example, improved efficiency, or a non-natural substrate, forexample, increased efficiency. For example, it has been reported thatthe enantioselectivity of a lipase from Pseudomonas aeruginosa wasimproved significantly (Reetz et al., Agnew. Chem. Int. Ed Engl.36:2830-2832 (1997)). This enzyme hydrolyzed p-nitrophenyl2-methyldecanoate with only 2% enantiomeric excess (ee) in favor of the(S)-acid. However, after four successive rounds of error-pronemutagenesis and screening, a variant was produced that catalyzed therequisite reaction with 81% ee (Reetz et al., Agnew. Chem. Int. Ed Engl.36:2830-2832 (1997)).

Directed evolution methods have been used to modify an enzyme tofunction on an array of non-natural substrates. The substratespecificity of the lipase in P. aeruginosa was broadened byrandomization of amino acid residues near the active site. This allowedfor the acceptance of alpha-substituted carboxylic acid esters by thisenzyme (Reetz et al., Agnew. Chem. Int. Ed Engl. 44:4192-4196 (2005)).In another successful modification of an enzyme, DNA shuffling wasemployed to create an Escherichia coli aminotransferase that acceptedβ-branched substrates, which were poorly accepted by the wild-typeenzyme (Yano et al., Proc. Nat. Acad. Sci. U.S.A. 95:5511-5515 (1998)).Specifically, at the end of four rounds of shuffling, the activity ofaspartate aminotransferase for valine and 2-oxovaline increased by up tofive orders of magnitude, while decreasing the activity towards thenatural substrate, aspartate, by up to 30-fold. Recently, an algorithmwas used to design a retro-aldolase that could be used to catalyze thecarbon-carbon bond cleavage in a non-natural and non-biologicalsubstrate, 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone (Jiang et al.,Science 319:1387-1391 (2008)). These algorithms used differentcombinations of four different catalytic motifs to design new enzyme,and 20 of the selected designs for experimental characterization hadfour-fold improved rates over the uncatalyzed reaction (Jiang et al.,Science 319:1387-1391 (2008)). Thus, not only are these engineeringapproaches capable of expanding the array of substrates on which anenzyme can act, but they allow the design and construction of veryefficient enzymes. For example, a method of DNA shuffling (randomchimeragenesis on transient templates or RACHITT) was reported to leadto an engineered monooxygenase that had an improved rate ofdesulfurization on complex substrates as well as 20-fold fasterconversion of a non-natural substrate (Coco et al., Nat. Biotechnol.19:354-359 (2001)). Similarly, the specific activity of a sluggishmutant triosephosphate isomerase enzyme was improved up to 19-fold from1.3 fold (Hermes et al., Proc. Nat. Acad. Sci. U.S.A. 87:696-700 1990)).This enhancement in specific activity was accomplished by using randommutagenesis over the whole length of the protein and the improvementcould be traced back to mutations in six amino acid residues.

The effectiveness of protein engineering approaches to alter thesubstrate specificity of an enzyme for a desired substrate has also beendemonstrated in several studies. Isopropylmalate dehydrogenase fromThermus thermophilus was modified by changing residues close to theactive site so that it could now act on malate and D-lactate assubstrates (Fujita et al., Biosci. Biotechnol. Biochem. 65:2695-2700(2001)). In this study as well as in others, it was pointed out that oneor a few residues could be modified to alter the substrate specificity.For example, the dihydroflavonol 4-reductase for which a single aminoacid was changed in the presumed substrate-binding region couldpreferentially reduce dihydrokaempferol (Johnson et al., Plant. J.25:325-333 (2001)). The substrate specificity of a very specificisocitrate dehydrogenase from Escherichia coli was changed formisocitrate to isopropylmalate by changing one residue in the active site(Doyle et al., Biochemistry 40:4234-4241 (2001)). Similarly, thecofactor specificity of a NAD⁺-dependent 1,5-hydroxyprostaglandindehydrogenase was altered to NADP⁺ by changing a few residues near theN-terminal end (Cho et al., Arch. Biochem. Biophys. 419:139-146 (2003)).Sequence analysis and molecular modeling analysis were used to identifythe key residues for modification, which were further studied bysite-directed mutagenesis.

Numerous examples exist spanning diverse classes of enzymes where thefunction of enzyme was changed to favor one non-natural substrate overthe natural substrate of the enzyme. A fucosidase was evolved from agalactosidase in E. coli by DNA shuffling and screening (Zhang et al.,Proc. Natl. Acad. Sci. U.S.A. 94:4504-4509 (1997)). Similarly, aspartateaminotransferase from E. coli was converted into a tyrosineaminotransferase using homology modeling and site-directed mutagenesis(Onuffer and Kirsch, Protein Sci., 4:1750-1757 (1995)). Site-directedmutagenesis of two residues in the active site of benzoylformatedecarboxylase from P. putida reportedly altered the affinity (K_(m))towards natural and non-natural substrates (Siegert et al., Protein EngDes Sel 18:345-357 (2005)). Cytochrome c peroxidase (CCP) fromSaccharomyces cerevisiae was subjected to directed molecular evolutionto generate mutants with increased activity against the classicalperoxidase substrate guaiacol, thus changing the substrate specificityof CCP from the protein cytochrome c to a small organic molecule. Afterthree rounds of DNA shuffling and screening, mutants were isolated whichpossessed a 300-fold increased activity against guaiacol and up to1000-fold increased specificity for this substrate relative to that forthe natural substrate (Iffland et al., Biochemistry 39:10790-10798(2000)).

In some cases, enzymes with different substrate preferences than eitherof the parent enzymes have been obtained. For example,biphenyl-dioxygenase-mediated degradation of polychlorinated biphenylswas improved by shuffling genes from two bacteria, Pseudomonaspseudoalcaligens and Burkholderia cepacia (Kumamaru et al., Nat.Biotechnol. 16:663-666 (1998)). The resulting chimeric biphenyloxygenases showed different substrate preferences than both the parentalenzymes and enhanced the degradation activity towards related biphenylcompounds and single aromatic ring hydrocarbons such as toluene andbenzene which were originally poor substrates for the enzyme.

In addition to changing enzyme specificity, it is also possible toenhance the activities on substrates for which the enzymes naturallyhave low activities. One study demonstrated that amino acid racemasefrom P. putida that had broad substrate specificity (on lysine,arginine, alanine, serine, methionine, cysteine, leucine and histidineamong others) but low activity towards tryptophan could be improvedsignificantly by random mutagenesis (Kino et al., Appl. Microbiol.Biotechnol. 73:1299-1305 (2007)). Similarly, the active site of thebovine BCKAD was engineered to favor alternate substrate acetyl-CoA(Meng and Chuang, Biochemistry 33:12879-12885 (1994)). An interestingaspect of these approaches is that even if random methods have beenapplied to generate these mutated enzymes with efficacious activities,the exact mutations or structural changes that confer the improvement inactivity can be identified. For example, in the aforementioned study,the mutations that facilitated improved activity on tryptophan wastraced back to two different positions.

Directed evolution has also been used to express proteins that aredifficult to express. For example, by subjecting horseradish peroxidaseto random mutagenesis and gene recombination, mutants were identifiedthat had more than 14-fold higher activity than the wild type (Lin etal., Biotechnol. Prog. 15:467-471 (1999)).

Another example of directed evolution shows the extensive modificationsto which an enzyme can be subjected to achieve a range of desiredfunctions. The enzyme lactate dehydrogenase from Bacillusstearothermophilus was subjected to site-directed mutagenesis, and threeamino acid substitutions were made at sites that were believed todetermine the specificity towards different hydroxyacids (Clarke et al.,Biochem. Biophys. Res. Commun. 148:15-23 (1987)). After these mutations,the specificity for oxaloacetate over pyruvate was increased to 500 incontrast to the wild type enzyme that had a catalytic specificity forpyruvate over oxaloacetate of 1000. This enzyme was further engineeredusing site-directed mutagenesis to have activity towards branched-chainsubstituted pyruvates (Wilks et al., Biochemistry 29:8587-8591 (1990)).Specifically, the enzyme had a 55-fold improvement in K_(cat) foralpha-ketoisocaproate. Three structural modifications were made in thesame enzyme to change its substrate specificity from lactate to malate.The enzyme was highly active and specific towards malate (Wilks et al.,Science 242:1541-1544 (1988)). The same enzyme from B.stearothermophilus was subsequently engineered to have high catalyticactivity towards alpha-keto acids with positively charged side chains,such as those containing ammonium groups (Hogan et al., Biochemistry34:4225-4230 (1995)). Mutants with acidic amino acids introduced atposition 102 of the enzyme favored binding of such side chain ammoniumgroups. The results obtained proved that the mutants showed up to25-fold improvements in k_(cat)/K_(m) values for omega-amino-alpha-ketoacid substrates. Interestingly, this enzyme was also structurallymodified to function as a phenyllactate dehydrogenase instead of alactate dehydrogenase (Wilks et al., Biochemistry 31:7802-7806 1992).Restriction sites were introduced into the gene for the enzyme whichallowede a region of the gene to be excised. This region coded for amobile surface loop of the polypeptide (residues 98-110) which normallyseals the active site from bulk solvent and is a major determinant ofsubstrate specificity. The variable length and sequence loops wereinserted so that hydroxyacid dehydrogenases with altered substratespecificities were generated. With one longer loop construction,activity with pyruvate was reduced one-million-fold but activity withphenylpyruvate was largely unaltered. A switch in specificity(k_(cat)/K_(m)) of 390,000-fold was achieved. The 1700:1 selectivity ofthis enzyme for phenylpyruvate over pyruvate is that required in aphenyllactate dehydrogenase. The studies described above indicate thatvarious approaches of enzyme engineering can be used to obtain enzymesfor the BDO pathways as disclosed herein.

As disclosed herein, biosynthetic pathways to 1,4-butanediol from anumber of central metabolic intermediates are can be utilized, includingacetyl-CoA, succinyl-CoA, alpha-ketoglutarate, glutamate,4-aminobutyrate, and homoserine. Acetyl-CoA, succinyl-CoA andalpha-ketoglutarate are common intermediates of the tricarboxylic acid(TCA) cycle, a series of reactions that is present in its entirety innearly all living cells that utilize oxygen for cellular respiration andis present in truncated forms in a number of anaerobic organisms.Glutamate is an amino acid that is derived from alpha-ketoglutarate viaglutamate dehydrogenase or any of a number of transamination reactions(see FIG. 8B). 4-aminobutyrate can be formed by the decarboxylation ofglutamate (see FIG. 8B) or from acetoacetyl-CoA via the pathwaydisclosed in FIG. 9C. Acetoacetyl-CoA is derived from the condensationof two acetyl-CoA molecules by way of the enzyme, acetyl-coenzyme Aacetyltransferase, or equivalently, acetoacetyl-coenzyme A thiolase.Homoserine is an intermediate in threonine and methionine metabolism,formed from oxaloacetate via aspartate. The conversion of oxaloacetateto homoserine requires one NADH, two NADPH, and one ATP.

Pathways other than those exemplified above also can be employed togenerate the biosynthesis of BDO in non-naturally occurring microbialorganisms. In one embodiment, biosynthesis can be achieved using aL-homoserine to BDO pathway (see FIG. 13). This pathway has a molaryield of 0.90 mol/mol glucose, which appears restricted by theavailability of reducing equivalents. A second pathway synthesizes BDOfrom acetoacetyl-CoA and is capable of achieving the maximum theoreticalyield of 1.091 mol/mol glucose (see FIG. 9). Implementation of eitherpathway can be achieved by introduction of two exogenous enzymes into ahost organism such as E. coli, and both pathways can additionallycomplement BDO production via succinyl-CoA. Pathway enzymes,thermodynamics, theoretical yields and overall feasibility are describedfurther below.

A homoserine pathway also can be engineered to generate BDO-producingmicrobial organisms. Homoserine is an intermediate in threonine andmethionine metabolism, formed from oxaloacetate via aspartate. Theconversion of oxaloacetate to homoserine requires one NADH, two NADPH,and one ATP (FIG. 2). Once formed, homoserine feeds into biosyntheticpathways for both threonine and methionine. In most organisms, highlevels of threonine or methionine feedback to repress the homoserinebiosynthesis pathway (Caspi et al., Nucleic Acids Res. 34:D511-D516(1990)).

The transformation of homoserine to 4-hydroxybutyrate (4-HB) can beaccomplished in two enzymatic steps as described herein. The first stepof this pathway is deamination of homoserine by a putative ammonialyase. In step 2, the product alkene, 4-hydroxybut-2-enoate is reducedto 4-HB by a putative reductase at the cost of one NADH. 4-HB can thenbe converted to BDO.

Enzymes available for catalyzing the above transformations are disclosedherein. For example, the ammonia lyase in step 1 of the pathway closelyresembles the chemistry of aspartate ammonia-lyase (aspartase).Aspartase is a widespread enzyme in microorganisms, and has beencharacterized extensively (Viola, R. E., Mol. Biol. 74:295-341 (2008)).The crystal structure of the E. coli aspartase has been solved (Shi etal., Biochemistry 36:9136-9144 (1997)), so it is therefore possible todirectly engineer mutations in the enzyme's active site that would alterits substrate specificity to include homoserine. The oxidoreductase instep 2 has chemistry similar to several well-characterized enzymesincluding fumarate reductase in the E. coli TCA cycle. Since thethermodynamics of this reaction are highly favorable, an endogenousreductase with broad substrate specificity will likely be able to reduce4-hydroxybut-2-enoate. The yield of this pathway under anaerobicconditions is 0.9 mol BDO per mol glucose.

The succinyl-CoA pathway was found to have a higher yield due to thefact that it is more energetically efficient. The conversion of oneoxaloacetate molecule to BDO via the homoserine pathway will require theexpenditure of 2 ATP equivalents. Because the conversion of glucose totwo oxaloacetate molecules can generate a maximum of 3 ATP moleculesassuming PEP carboxykinase to be reversible, the overall conversion ofglucose to BDO via homoserine has a negative energetic yield. Asexpected, if it is assumed that energy can be generated via respiration,the maximum yield of the homoserine pathway increases to 1.05 mol/molglucose which is 96% of the succinyl-CoA pathway yield. The succinyl-CoApathway can channel some of the carbon flux through pyruvatedehydrogenase and the oxidative branch of the TCA cycle to generate bothreducing equivalents and succinyl-CoA without an energetic expenditure.Thus, it does not encounter the same energetic difficulties as thehomoserine pathway because not all of the flux is channeled throughoxaloacetate to succinyl-CoA to BDO. Overall, the homoserine pathwaydemonstrates a high-yielding route to BDO.

An acetoacetate pathway also can be engineered to generate BDO-producingmicrobial organisms. Acetoacetate can be formed from acetyl-CoA byenzymes involved in fatty acid metabolism, including acetyl-CoAacetyltransferase and acetoacetyl-CoA transferase. Biosynthetic routesthrough acetoacetate are also particularly useful in microbial organismsthat can metabolize single carbon compounds such as carbon monoxide,carbon dioxide or methanol to form acetyl-CoA.

A three step route from acetoacetyl-CoA to 4-aminobutyrate (see FIG. 9C)can be used to synthesize BDO through acetoacetyl-CoA. 4-Aminobutyratecan be converted to succinic semialdehyde as shown in FIG. 8B. Succinicsemialdehyde, which is one reduction step removed from succinyl-CoA orone decarboxylation step removed from α-ketoglutarate, can be convertedto BDO following three reductions steps (FIG. 1). Briefly, step 1 ofthis pathway involves the conversion of acetoacetyl-CoA to acetoacetateby, for example, the E. coli acetoacetyl-CoA transferase encoded by theatoA and atoD genes (Hanai et al., Appl. Environ. Microbiol. 73:7814-7818 (2007)). Step 2 of the acetoacetyl-CoA biopathway entailsconversion of acetoacetate to 3-aminobutanoate by an ω-aminotransferase.The ω-amino acid:pyruvate aminotransferase (w-APT) from Alcaligensdenitrificans was overexpressed in E. coli and shown to have a highactivity toward 3-aminobutanoate in vitro (Yun et al., Appl. Environ.Microbiol. 70:2529-2534 (2004)).

In step 2, a putative aminomutase shifts the amine group from the 3- tothe 4-position of the carbon backbone. An aminomutase performing thisfunction on 3-aminobutanoate has not been characterized, but an enzymefrom Clostridium sticklandii has a very similar mechanism. The enzyme,D-lysine-5,6-aminomutase, is involved in lysine biosynthesis.

The synthetic route to BDO from acetoacetyl-CoA passes through4-aminobutanoate, a metabolite in E. coli that is normally formed fromdecarboxylation of glutamate. Once formed, 4-aminobutanoate can beconverted to succinic semialdehyde by 4-aminobutanoate transaminase(2.6.1.19), an enzyme which has been biochemically characterized.

One consideration for selecting candidate enzymes in this pathway is thestereoselectivity of the enzymes involved in steps 2 and 3. The ω-ABT inAlcaligens denitrificans is specific to the L-stereoisomer of3-aminobutanoate, while D-lysine-5,6-aminomutase likely requires theD-stereoisomer. If enzymes with complementary stereoselectivity are notinitially found or engineered, a third enzyme can be added to thepathway with racemase activity that can convert L-3-aminobutanoate toD-3-aminobutanoate. While amino acid racemases are widespread, whetherthese enzymes can function on ω-amino acids is not known.

The maximum theoretical molar yield of this pathway under anaerobicconditions is 1.091 mol/mol glucose. In order to generate flux fromacetoacetyl-CoA to BDO it was necessary to assume thatacetyl-CoA:acetoacetyl-CoA transferase is reversible. The function ofthis enzyme in E. coli is to metabolize short-chain fatty acids by firstconverting them into thioesters.

While the operation of acetyl-CoA:acetoacetyl-CoA transferase in theacetate-consuming direction has not been demonstrated experimentally inE. coli, studies on similar enzymes in other organisms support theassumption that this reaction is reversible. The enzymebutyryl-CoA:acetate:CoA transferase in gut microbes Roseburia sp. and F.prasnitzii operates in the acetate utilizing direction to producebutyrate (Duncan et al., Appl. Environ. Microbiol. 68:5186-5190 (2002)).Another very similar enzyme, acetyl:succinate CoA-transferase inTrypanosoma brucei, also operates in the acetate utilizing direction.This reaction has a Δ_(rxn)G close to equilibrium, so highconcentrations of acetate can likely drive the reaction in the directionof interest. At the maximum theoretical BDO production rate of 1.09mol/mol glucose simulations predict that E. coli can generate 1.098 molATP per mol glucose with no fermentation byproducts. This ATP yieldshould be sufficient for cell growth, maintenance, and production. Theacetoacetatyl-CoA biopathway is a high-yielding route to BDO fromacetyl-CoA.

Therefore, in addition to any of the various modifications exemplifiedpreviously for establishing 4-HB biosynthesis in a selected host, theBDO producing microbial organisms can include any of the previouscombinations and permutations of 4-HB pathway metabolic modifications aswell as any combination of expression for CoA-independent aldehydedehydrogenase, CoA-dependent aldehyde dehydrogenase or an alcoholdehydrogenase or other enzymes disclosed herein to generate biosyntheticpathways for GBL and/or BDO. Therefore, the BDO producers of theinvention can have exogenous expression of, for example, one, two,three, four, five, six, seven, eight, nine, or up to all enzymescorresponding to any of the 4-HB pathway and/or any of the BDO pathwayenzymes disclosed herein.

Design and construction of the genetically modified microbial organismsis carried out using methods well known in the art to achieve sufficientamounts of expression to produce BDO. In particular, the non-naturallyoccurring microbial organisms of the invention can achieve biosynthesisof BDO resulting in intracellular concentrations between about 0.1-200mM or more, such as about 0.1-25 mM or more, as discussed above. Forexample, the intracellular concentration of BDO is between about 3-20mM, particularly between about 5-15 mM and more particularly betweenabout 8-12 mM, including about 10 mM or more. Intracellularconcentrations between and above each of these exemplary ranges also canbe achieved from the non-naturally occurring microbial organisms of theinvention. As with the 4-HB producers, the BDO producers also can besustained, cultured or fermented under anaerobic conditions.

The invention further provides a method for the production of 4-HB. Themethod includes culturing a non-naturally occurring microbial organismhaving a 4-hydroxybutanoic acid (4-HB) biosynthetic pathway comprisingat least one exogenous nucleic acid encoding 4-hydroxybutanoatedehydrogenase, CoA-independent succinic semialdehyde dehydrogenase,succinyl-CoA synthetase, CoA-dependent succinic semialdehydedehydrogenase, glutamate:succinic semialdehyde transaminase,α-ketoglutarate decarboxylase, or glutamate decarboxylase undersubstantially anaerobic conditions for a sufficient period of time toproduce monomeric 4-hydroxybutanoic acid (4-HB). The method canadditionally include chemical conversion of 4-HB to GBL and to BDO orTHF, for example.

Additionally provided is a method for the production of 4-HB. The methodincludes culturing a non-naturally occurring microbial organism having a4-hydroxybutanoic acid (4-HB) biosynthetic pathway including at leastone exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase,succinyl-CoA synthetase, CoA-dependent succinic semialdehydedehydrogenase or α-ketoglutarate decarboxylase under substantiallyanaerobic conditions for a sufficient period of time to producemonomeric 4-hydroxybutanoic acid (4-HB). The 4-HB product can besecreted into the culture medium.

Further provided is a method for the production of BDO. The methodincludes culturing a non-naturally occurring microbial biocatalyst ormicrobial organism, comprising a microbial organism having4-hydroxybutanoic acid (4-HB) and 1,4-butanediol (BDO) biosyntheticpathways, the pathways including at least one exogenous nucleic acidencoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase,CoA-dependent succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:CoAtransferase, 4-hydroxybutyrate kinase, phosphotranshydroxybutyrylase,α-ketoglutarate decarboxylase, aldehyde dehydrogenase, alcoholdehydrogenase or an aldehyde/alcohol dehydrogenase for a sufficientperiod of time to produce 1,4-butanediol (BDO). The BDO product can besecreted into the culture medium.

Additionally provided are methods for producing BDO by culturing anon-naturally occurring microbial organism having a BDO pathway of theinvention. The BDO pathway can comprise at least one exogenous nucleicacid encoding a BDO pathway enzyme expressed in a sufficient amount toproduce BDO, under conditions and for a sufficient period of time toproduce BDO, the BDO pathway comprising 4-aminobutyrate CoA transferase,4-aminobutyryl-CoA hydrolase, 4-aminobutyrate-CoA ligase,4-aminobutyryl-CoA oxidoreductase (deaminating), 4-aminobutyryl-CoAtransaminase, or 4-hydroxybutyryl-CoA dehydrogenase (see Example VII andTable 17).

Alternatively, the BDO pathway can comprise at least one exogenousnucleic acid encoding a BDO pathway enzyme expressed in a sufficientamount to produce BDO, under conditions and for a sufficient period oftime to produce BDO, the BDO pathway comprising 4-aminobutyrate CoAtransferase, 4-aminobutyryl-CoA hydrolase, 4-aminobutyrate-CoA ligase,4-aminobutyryl-CoA reductase (alcohol forming), 4-aminobutyryl-CoAreductase, 4-aminobutan-1-ol dehydrogenase, 4-aminobutan-1-oloxidoreductase (deaminating) or 4-aminobutan-1-ol transaminase (seeExample VII and Table 18).

In addition, the invention provides a method for producing BDO,comprising culturing a non-naturally occurring microbial organism havinga BDO pathway, the pathway comprising at least one exogenous nucleicacid encoding a BDO pathway enzyme expressed in a sufficient amount toproduce BDO, under conditions and for a sufficient period of time toproduce BDO, the BDO pathway comprising 4-aminobutyrate kinase,4-aminobutyraldehyde dehydrogenase (phosphorylating), 4-aminobutan-1-oldehydrogenase, 4-aminobutan-1-oloxidoreductase (deaminating),4-aminobutan-1-ol transaminase, [(4-aminobutanolyl)oxy]phosphonic acidoxidoreductase (deaminating), [(4-aminobutanolyl)oxy]phosphonic acidtransaminase, 4-hydroxybutyryl-phosphate dehydrogenase, or4-hydroxybutyraldehyde dehydrogenase (phosphorylating) (see Example VIIand Table 19).

The invention further provides a method for producing BDO, comprisingculturing a non-naturally occurring microbial organism having a BDOpathway, the pathway comprising at least one exogenous nucleic acidencoding a BDO pathway enzyme expressed in a sufficient amount toproduce BDO, under conditions and for a sufficient period of time toproduce BDO, the BDO pathway comprising alpha-ketoglutarate 5-kinase,2,5-dioxopentanoic semialdehyde dehydrogenase (phosphorylating),2,5-dioxopentanoic acid reductase, alpha-ketoglutarate CoA transferase,alpha-ketoglutaryl-CoA hydrolase, alpha-ketoglutaryl-CoA ligase,alpha-ketoglutaryl-CoA reductase, 5-hydroxy-2-oxopentanoic aciddehydrogenase, alpha-ketoglutaryl-CoA reductase (alcohol forming),5-hydroxy-2-oxopentanoic acid decarboxylase, or 5-hydroxy-2-oxopentanoicacid dehydrogenase (decarboxylation) (see Example VIII and Table 20).

The invention additionally provides a method for producing BDO,comprising culturing a non-naturally occurring microbial organism havinga BDO pathway, the pathway comprising at least one exogenous nucleicacid encoding a BDO pathway enzyme expressed in a sufficient amount toproduce BDO, under conditions and for a sufficient period of time toproduce BDO, the BDO pathway comprising glutamate CoA transferase,glutamyl-CoA hydrolase, glutamyl-CoA ligase, glutamate 5-kinase,glutamate-5-semialdehyde dehydrogenase (phosphorylating), glutamyl-CoAreductase, glutamate-5-semialdehyde reductase, glutamyl-CoA reductase(alcohol forming), 2-amino-5-hydroxypentanoic acid oxidoreductase(deaminating), 2-amino-5-hydroxypentanoic acid transaminase,5-hydroxy-2-oxopentanoic acid decarboxylase, 5-hydroxy-2-oxopentanoicacid dehydrogenase (decarboxylation) (see Example IX and Table 21).

The invention additionally includes a method for producing BDO,comprising culturing a non-naturally occurring microbial organism havinga BDO pathway, the pathway comprising at least one exogenous nucleicacid encoding a BDO pathway enzyme expressed in a sufficient amount toproduce BDO, under conditions and for a sufficient period of time toproduce BDO, the BDO pathway comprising 3-hydroxybutyryl-CoAdehydrogenase, 3-hydroxybutyryl-CoA dehydratase, vinylacetyl-CoAΔ-isomerase, or 4-hydroxybutyryl-CoA dehydratase (see Example X andTable 22).

Also provided is a method for producing BDO, comprising culturing anon-naturally occurring microbial organism having a BDO pathway, thepathway comprising at least one exogenous nucleic acid encoding a BDOpathway enzyme expressed in a sufficient amount to produce BDO, underconditions and for a sufficient period of time to produce BDO, the BDOpathway comprising homoserine deaminase, homoserine CoA transferase,homoserine-CoA hydrolase, homoserine-CoA ligase, homoserine-CoAdeaminase, 4-hydroxybut-2-enoyl-CoA transferase,4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA ligase,4-hydroxybut-2-enoate reductase, 4-hydroxybutyryl-CoA transferase,4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or4-hydroxybut-2-enoyl-CoA reductase (see Example XI and Table 23).

The invention additionally provides a method for producing BDO,comprising culturing a non-naturally occurring microbial organism havinga BDO pathway, the pathway comprising at least one exogenous nucleicacid encoding a BDO pathway enzyme expressed in a sufficient amount toproduce BDO, under conditions and for a sufficient period of time toproduce BDO, the BDO pathway comprising succinyl-CoA reductase (alcoholforming), 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase,4-hydroxybutanal dehydrogenase (phosphorylating). Such a BDO pathway canfurther comprise succinyl-CoA reductase, 4-hydroxybutyratedehydrogenase, 4-hydroxybutyryl-CoA transferase, 4-hydroxybutyratekinase, phosphotrans-4-hydroxybutyrylase, 4-hydroxybutyryl-CoAreductase, 4-hydroxybutyryl-CoA reductase (alcohol forming), or1,4-butanediol dehydrogenase.

Also provided is a method for producing BDO, comprising culturing anon-naturally occurring microbial organism having a BDO pathway, thepathway comprising at least one exogenous nucleic acid encoding a BDOpathway enzyme expressed in a sufficient amount to produce BDO, underconditions and for a sufficient period of time to produce BDO, the BDOpathway comprising glutamate dehydrogenase, 4-aminobutyrateoxidoreductase (deaminating), 4-aminobutyrate transaminase, glutamatedecarboxylase, 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoAligase, 4-hydroxybutanal dehydrogenase (phosphorylating).

The invention additionally provides methods of producing a desiredproduct using the genetically modified organisms disclosed herein thatallow improved production of a desired product such as BDO by increasingthe product or decreasing undesirable byproducts. Thus, the inventionprovides a method for producing 1,4-butanediol (BDO), comprisingculturing the non-naturally occurring microbial organisms disclosedherein under conditions and for a sufficient period of time to produceBDO. In one embodiment, the invention provides a method of producing BDOusing a non-naturally occurring microbial organism, comprising amicrobial organism having a 1,4-butanediol (BDO) pathway comprising atleast one exogenous nucleic acid encoding a BDO pathway enzyme expressedin a sufficient amount to produce BDO. In one embodiment, the microbialorganism is genetically modified to express exogenous succinyl-CoAsynthetase (see Example XII). For example, the succinyl-CoA synthetasecan be encoded by an Escherichia coli sucCD genes.

In another embodiment, the microbial organism is genetically modified toexpress exogenous alpha-ketoglutarate decarboxylase (see Example XIII).For example, the alpha-ketoglutarate decarboxylase can be encoded by theMycobacterium bovis sucA gene. In still another embodiment, themicrobial organism is genetically modified to express exogenoussuccinate semialdehyde dehydrogenase and 4-hydroxybutyrate dehydrogenaseand optionally 4-hydroxybutyryl-CoA/acetyl-CoA transferase (see ExampleXIII). For example, the succinate semialdehyde dehydrogenase(CoA-dependent), 4-hydroxybutyrate dehydrogenase and4-hydroxybutyryl-CoA/acetyl-CoA transferase can be encoded byPorphyromonas gingivalis W83 genes. In an additional embodiment, themicrobial organism is genetically modified to express exogenous butyratekinase and phosphotransbutyrylase (see Example XIII). For example, thebutyrate kinase and phosphotransbutyrylase can be encoded by Clostridiumacetobutilicum buk1 and ptb genes.

In yet another embodiment, the microbial organism is geneticallymodified to express exogenous 4-hydroxybutyryl-CoA reductase (seeExample XIII). For example, the 4-hydroxybutyryl-CoA reductase can beencoded by Clostridium beijerinckii ald gene. Additionally, in anembodiment of the invention, the microbial organism is geneticallymodified to express exogenous 4-hydroxybutanal reductase (see ExampleXIII). For example, the 4-hydroxybutanal reductase can be encoded byGeobacillus thermoglucosidasius adh1 gene. In another embodiment, themicrobial organism is genetically modified to express exogenous pyruvatedehydrogenase subunits (see Example XIV). For example, the exogenouspyruvate dehydrogenase can be NADH insensitive. The pyruvatedehydrogenase subunit can be encoded by the Klebsiella pneumonia lpdAgene. In a particular embodiment, the pyruvate dehydrogenase subunitgenes of the microbial organism can be under the control of a pyruvateformate lyase promoter.

In still another embodiment, the microbial organism is geneticallymodified to disrupt a gene encoding an aerobic respiratory controlregulatory system (see Example XV). For example, the disruption can beof the arcA gene. Such an organism can further comprise disruption of agene encoding malate dehydrogenase. In a further embodiment, themicrobial organism is genetically modified to express an exogenous NADHinsensitive citrate synthase (see Example XV). For example, the NADHinsensitive citrate synthase can be encoded by gltA, such as an R163Lmutant of gltA. In still another embodiment, the microbial organism isgenetically modified to express exogenous phosphoenolpyruvatecarboxykinase (see Example XVI). For example, the phosphoenolpyruvatecarboxykinase can be encoded by an Haemophilus influenzaphosphoenolpyruvate carboxykinase gene. It is understood that strainsexemplified herein for improved production of BDO can similarly be used,with appropriate modifications, to produce other desired products, forexample, 4-hydroxybutyrate or other desired products disclosed herein.

The invention additionally provides a method for producing4-hydroxybutanal by culturing a non-naturally occurring microbialorganism, comprising a 4-hydroxybutanal pathway comprising at least oneexogenous nucleic acid encoding a 4-hydroxybutanal pathway enzymeexpressed in a sufficient amount to produce 4-hydroxybutanal, the4-hydroxybutanal pathway comprising succinyl-CoA reductase (aldehydeforming); 4-hydroxybutyrate dehydrogenase; and 4-hydroxybutyratereductase (see FIG. 58, steps A-C-D). The invention also provides amethod for producing 4-hydroxybutanal by culturing a non-naturallyoccurring microbial organism, comprising a 4-hydroxybutanal pathwaycomprising at least one exogenous nucleic acid encoding a4-hydroxybutanal pathway enzyme expressed in a sufficient amount toproduce 4-hydroxybutanal, the 4-hydroxybutanal pathway comprisingalpha-ketoglutarate decarboxylase; 4-hydroxybutyrate dehydrogenase; and4-hydroxybutyrate reductase (FIG. 58, steps B-C-D).

The invention further provides a method for producing 4-hydroxybutanalby culturing a non-naturally occurring microbial organism, comprising a4-hydroxybutanal pathway comprising at least one exogenous nucleic acidencoding a 4-hydroxybutanal pathway enzyme expressed in a sufficientamount to produce 4-hydroxybutanal, the 4-hydroxybutanal pathwaycomprising succinate reductase; 4-hydroxybutyrate dehydrogenase, and4-hydroxybutyrate reductase (see FIG. 62, steps F-C-D). In yet anotherembodiment, the invention provides a method for producing4-hydroxybutanal by culturing a non-naturally occurring microbialorganism, comprising a 4-hydroxybutanal pathway comprising at least oneexogenous nucleic acid encoding a 4-hydroxybutanal pathway enzymeexpressed in a sufficient amount to produce 4-hydroxybutanal, the4-hydroxybutanal pathway comprising alpha-ketoglutarate decarboxylase,or glutamate dehydrogenase or glutamate transaminase and glutamatedecarboxylase and 4-aminobutyrate dehydrogenase or 4-aminobutyratetransaminase; 4-hydroxybutyrate dehydrogenase; and 4-hydroxybutyratereductase (see FIG. 62, steps B or ((J or K)-L-(M or N))-C-D).

The invention also provides a method for producing 4-hydroxybutanal byculturing a non-naturally occurring microbial organism, comprising a4-hydroxybutanal pathway comprising at least one exogenous nucleic acidencoding a 4-hydroxybutanal pathway enzyme expressed in a sufficientamount to produce 4-hydroxybutanal, the 4-hydroxybutanal pathwaycomprising alpha-ketoglutarate reductase; 5-hydroxy-2-oxopentanoatedehydrogenase; and 5-hydroxy-2-oxopentanoate decarboxylase (see FIG. 62,steps X-Y-Z). The invention further provides a method for producing4-hydroxybutyryl-CoA by culturing a non-naturally occurring microbialorganism, comprising a 4-hydroxybutyryl-CoA pathway comprising at leastone exogenous nucleic acid encoding a 4-hydroxybutyryl-CoA pathwayenzyme expressed in a sufficient amount to produce 4-hydroxybutyryl-CoA,the 4-hydroxybutyryl-CoA pathway comprising alpha-ketoglutaratereductase; 5-hydroxy-2-oxopentanoate dehydrogenase; and5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation) (see FIG. 62,steps X-Y-AA).

The invention additionally provides a method for producing putrescine byculturing a non-naturally occurring microbial organism, comprising aputrescine pathway comprising at least one exogenous nucleic acidencoding a putrescine pathway enzyme expressed in a sufficient amount toproduce putrescine, the putrescine pathway comprising succinatereductase; 4-aminobutyrate dehydrogenase or 4-aminobutyratetransaminase; 4-aminobutyrate reductase; and putrescine dehydrogenase orputrescine transaminase (see FIG. 63, steps F-M/N-C-D/E). In stillanother embodiment, the invention provides a method for producingputrescine by culturing a non-naturally occurring microbial organism,comprising a putrescine pathway comprising at least one exogenousnucleic acid encoding a putrescine pathway enzyme expressed in asufficient amount to produce putrescine, the putrescine pathwaycomprising alpha-ketoglutarate decarboxylase; 4-aminobutyratedehydrogenase or 4-aminobutyrate transaminase; 4-aminobutyratereductase; and putrescine dehydrogenase or putrescine transaminase (seeFIG. 63, steps B-M/N-C-D/E). The invention additionally provides amethod for producing putrescine by culturing a non-naturally occurringmicrobial organism, comprising a putrescine pathway comprising at leastone exogenous nucleic acid encoding a putrescine pathway enzymeexpressed in a sufficient amount to produce putrescine, the putrescinepathway comprising glutamate dehydrogenase or glutamate transaminase;glutamate decarboxylase; 4-aminobutyrate reductase; and putrescinedehydrogenase or putrescine transaminase (see FIG. 63, stepsJ/K-L-C-D/E).

The invention provides in another embodiment a method for producingputrescine by culturing a non-naturally occurring microbial organism,comprising a putrescine pathway comprising at least one exogenousnucleic acid encoding a putrescine pathway enzyme expressed in asufficient amount to produce putrescine, the putrescine pathwaycomprising alpha-ketoglutarate reductase; 5-amino-2-oxopentanoatedehydrogenase or 5-amino-2-oxopentanoate transaminase;5-amino-2-oxopentanoate decarboxylase; and putrescine dehydrogenase orputrescine transaminase (see FIG. 63, steps O-P/Q-R-D/E). Also providedis a method for producing putrescine by culturing a non-naturallyoccurring microbial organism, comprising a putrescine pathway comprisingat least one exogenous nucleic acid encoding a putrescine pathway enzymeexpressed in a sufficient amount to produce putrescine, the putrescinepathway comprising alpha-ketoglutarate reductase;5-amino-2-oxopentanoate dehydrogenase or 5-amino-2-oxopentanoatetransaminase; ornithine dehydrogenase or ornithine transaminase; andornithine decarboxylase (see FIG. 63, steps O-P/Q-S/T-U). It isunderstood that a microbial organism comprising any of the pathwaysdisclosed herein can be used to produce a a desired product orintermediate, including 4-HB, 4-HBal, BDO or putrescine.

It is understood that, in methods of the invention, any of the one ormore exogenous nucleic acids can be introduced into a microbial organismto produce a non-naturally occurring microbial organism of theinvention. The nucleic acids can be introduced so as to confer, forexample, a 4-HB, BDO, THF or GBL biosynthetic pathway onto the microbialorganism. Alternatively, encoding nucleic acids can be introduced toproduce an intermediate microbial organism having the biosyntheticcapability to catalyze some of the required reactions to confer 4-HB,BDO, THF or GBL biosynthetic capability. For example, a non-naturallyoccurring microbial organism having a 4-HB biosynthetic pathway cancomprise at least two exogenous nucleic acids encoding desired enzymes,such as the combination of 4-hydroxybutanoate dehydrogenase andα-ketoglutarate decarboxylase; 4-hydroxybutanoate dehydrogenase andCoA-independent succinic semialdehyde dehydrogenase; 4-hydroxybutanoatedehydrogenase and CoA-dependent succinic semialdehyde dehydrogenase;CoA-dependent succinic semialdehyde dehydrogenase and succinyl-CoAsynthetase; succinyl-CoA synthetase and glutamate decarboxylase, and thelike. Thus, it is understood that any combination of two or more enzymesof a biosynthetic pathway can be included in a non-naturally occurringmicrobial organism of the invention. Similarly, it is understood thatany combination of three or more enzymes of a biosynthetic pathway canbe included in a non-naturally occurring microbial organism of theinvention, for example, 4-hydroxybutanoate dehydrogenase,α-ketoglutarate decarboxylase and CoA-dependent succinic semialdehydedehydrogenase; CoA-independent succinic semialdehyde dehydrogenase andsuccinyl-CoA synthetase; 4-hydroxybutanoate dehydrogenase, CoA-dependentsuccinic semialdehyde dehydrogenase and glutamate:succinic semialdehydetransaminase, and so forth, as desired, so long as the combination ofenzymes of the desired biosynthetic pathway results in production of thecorresponding desired product.

Similarly, for example, with respect to any one or more exogenousnucleic acids introduced to confer BDO production, a non-naturallyoccurring microbial organism having a BDO biosynthetic pathway cancomprise at least two exogenous nucleic acids encoding desired enzymes,such as the combination of 4-hydroxybutanoate dehydrogenase andα-ketoglutarate decarboxylase; 4-hydroxybutanoate dehydrogenase and4-hydroxybutyryl CoA:acetyl-CoA transferase; 4-hydroxybutanoatedehydrogenase and butyrate kinase; 4-hydroxybutanoate dehydrogenase andphosphotransbutyrylase; 4-hydroxybutyryl CoA:acetyl-CoA transferase andaldehyde dehydrogenase; 4-hydroxybutyryl CoA:acetyl-CoA transferase andalcohol dehydrogenase; 4-hydroxybutyryl CoA:acetyl-CoA transferase andan aldehyde/alcohol dehydrogenase, 4-aminobutyrate-CoA transferase and4-aminobutyryl-CoA transaminase; 4-aminobutyrate kinase and4-aminobutan-1-ol oxidoreductase (deaminating), and the like. Thus, itis understood that any combination of two or more enzymes of abiosynthetic pathway can be included in a non-naturally occurringmicrobial organism of the invention. Similarly, it is understood thatany combination of three or more enzymes of a biosynthetic pathway canbe included in a non-naturally occurring microbial organism of theinvention, for example, 4-hydroxybutanoate dehydrogenase,α-ketoglutarate decarboxylase and 4-hydroxybutyryl CoA:acetyl-CoAtransferase; 4-hydroxybutanoate dehydrogenase, butyrate kinase andphosphotransbutyrylase; 4-hydroxybutanoate dehydrogenase,4-hydroxybutyryl CoA:acetyl-CoA transferase and aldehyde dehydrogenase;4-hydroxybutyryl CoA:acetyl-CoA transferase, aldehyde dehydrogenase andalcohol dehydrogenase; butyrate kinase, phosphotransbutyrylase and analdehyde/alcohol dehydrogenase; 4-aminobutyryl-CoA hydrolase,4-aminobutyryl-CoA reductase and 4-amino butan-1-ol transaminase;3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase and4-hydroxybutyryl-CoA dehydratase, and the like. Similarly, anycombination of four, five or more enzymes of a biosynthetic pathway asdisclosed herein can be included in a non-naturally occurring microbialorganism of the invention, as desired, so long as the combination ofenzymes of the desired biosynthetic pathway results in production of thecorresponding desired product.

Any of the non-naturally occurring microbial organisms described hereincan be cultured to produce and/or secrete the biosynthetic products ofthe invention. For example, the 4-HB producers can be cultured for thebiosynthetic production of 4-HB. The 4-HB can be isolated or be treatedas described below to generate GBL, THF and/or BDO. Similarly, the BDOproducers can be cultured for the biosynthetic production of BDO. TheBDO can be isolated or subjected to further treatments for the chemicalsynthesis of BDO family compounds, as disclosed herein.

The growth medium can include, for example, any carbohydrate sourcewhich can supply a source of carbon to the non-naturally occurringmicroorganism. Such sources include, for example, sugars such asglucose, sucrose, xylose, arabinose, galactose, mannose, fructose andstarch. Other sources of carbohydrate include, for example, renewablefeedstocks and biomass. Exemplary types of biomasses that can be used asfeedstocks in the methods of the invention include cellulosic biomass,hemicellulosic biomass and lignin feedstocks or portions of feedstocks.Such biomass feedstocks contain, for example, carbohydrate substratesuseful as carbon sources such as glucose, sucrose, xylose, arabinose,galactose, mannose, fructose and starch. Given the teachings andguidance provided herein, those skilled in the art will understand thatrenewable feedstocks and biomass other than those exemplified above alsocan be used for culturing the microbial organisms of the invention forthe production of 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine and othercompounds of the invention.

Accordingly, given the teachings and guidance provided herein, thoseskilled in the art will understand that a non-naturally occurringmicrobial organism can be produced that secretes the biosynthesizedcompounds of the invention when grown on a carbon source such as acarbohydrate. Such compounds include, for example, 4-HB, 4-HBal,4-HBCoA, BDO or putrescine and any of the intermediates metabolites inthe 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine pathways and/or thecombined 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine pathways. All that isrequired is to engineer in one or more of the enzyme activities shown inFIG. 1 to achieve biosynthesis of the desired compound or intermediateincluding, for example, inclusion of some or all of the 4-HB, 4-HBal,4-HBCoA, BDO or putrescine biosynthetic pathways. Accordingly, theinvention provides a non-naturally occurring microbial organism thatsecretes 4-HB when grown on a carbohydrate, secretes BDO when grown on acarbohydrate and/or secretes any of the intermediate metabolites shownin FIG. 1, 8-13, 58, 62 or 63 when grown on a carbohydrate. A BDOproducing microbial organisms of the invention can initiate synthesisfrom, for example, succinate, succinyl-CoA, α-ketogluterate, succinicsemialdehyde, 4-HB, 4-hydroxybutyrylphosphate, 4-hydroxybutyryl-CoA(4-HB-CoA) and/or 4-hydroxybutyraldehyde.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described below in the Examples. Any of these conditions can beemployed with the non-naturally occurring microbial organisms as well asother anaerobic conditions well known in the art. Under such anaerobicconditions, the 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine producers cansynthesize monomeric 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine,respectively, at intracellular concentrations of 5-10 mM or more as wellas all other concentrations exemplified previously.

A number of downstream compounds also can be generated for the 4-HB,4-HBal, 4-HBCoA, BDO or putrescine producing non-naturally occurringmicrobial organisms of the invention. With respect to the 4-HB producingmicrobial organisms of the invention, monomeric 4-HB and GBL exist inequilibrium in the culture medium. The conversion of 4-HB to GBL can beefficiently accomplished by, for example, culturing the microbialorganisms in acid pH medium. A pH less than or equal to 7.5, inparticular at or below pH 5.5, spontaneously converts 4-HB to GBL.

The resultant GBL can be separated from 4-HB and other components in theculture using a variety of methods well known in the art. Suchseparation methods include, for example, the extraction proceduresexemplified in the Examples as well as methods which include continuousliquid-liquid extraction, pervaporation, membrane filtration, membraneseparation, reverse osmosis, electrodialysis, distillation,crystallization, centrifugation, extractive filtration, ion exchangechromatography, size exclusion chromatography, adsorptionchromatography, and ultrafiltration. All of the above methods are wellknown in the art. Separated GBL can be further purified by, for example,distillation.

Another down stream compound that can be produced from the 4-HBproducing non-naturally occurring microbial organisms of the inventionincludes, for example, BDO. This compound can be synthesized by, forexample, chemical hydrogenation of GBL. Chemical hydrogenation reactionsare well known in the art. One exemplary procedure includes the chemicalreduction of 4-HB and/or GBL or a mixture of these two componentsderiving from the culture using a heterogeneous or homogeneoushydrogenation catalyst together with hydrogen, or a hydride-basedreducing agent used stoichiometrically or catalytically, to produce1,4-butanediol.

Other procedures well known in the art are equally applicable for theabove chemical reaction and include, for example, WO No. 82/03854(Bradley, et al.), which describes the hydrogenolysis ofgamma-butyrolactone in the vapor phase over a copper oxide and zincoxide catalyst. British Pat. No. 1,230,276, which describes thehydrogenation of gamma-butyrolactone using a copper oxide-chromium oxidecatalyst. The hydrogenation is carried out in the liquid phase. Batchreactions also are exemplified having high total reactor pressures.Reactant and product partial pressures in the reactors are well abovethe respective dew points. British Pat. No. 1,314,126, which describesthe hydrogenation of gamma-butyrolactone in the liquid phase over anickel-cobalt-thorium oxide catalyst. Batch reactions are exemplified ashaving high total pressures and component partial pressures well aboverespective component dew points. British Pat. No. 1,344,557, whichdescribes the hydrogenation of gamma-butyrolactone in the liquid phaseover a copper oxide-chromium oxide catalyst. A vapor phase orvapor-containing mixed phase is indicated as suitable in some instances.A continuous flow tubular reactor is exemplified using high totalreactor pressures. British Pat. No. 1,512,751, which describes thehydrogenation of gamma-butyrolactone to 1,4-butanediol in the liquidphase over a copper oxide-chromium oxide catalyst. Batch reactions areexemplified with high total reactor pressures and, where determinable,reactant and product partial pressures well above the respective dewpoints. U.S. Pat. No. 4,301,077, which describes the hydrogenation to1,4-butanediol of gamma-butyrolactone over a Ru—Ni—Co—Zn catalyst. Thereaction can be conducted in the liquid or gas phase or in a mixedliquid-gas phase. Exemplified are continuous flow liquid phase reactionsat high total reactor pressures and relatively low reactorproductivities. U.S. Pat. No. 4,048,196, which describes the productionof 1,4-butanediol by the liquid phase hydrogenation ofgamma-butyrolactone over a copper oxide-zinc oxide catalyst. Furtherexemplified is a continuous flow tubular reactor operating at high totalreactor pressures and high reactant and product partial pressures. AndU.S. Pat. No. 4,652,685, which describes the hydrogenation of lactonesto glycols.

A further downstream compound that can be produced form the 4-HBproducing microbial organisms of the invention includes, for example,THF. This compound can be synthesized by, for example, chemicalhydrogenation of GBL. One exemplary procedure well known in the artapplicable for the conversion of GBL to THF includes, for example,chemical reduction of 4-HB and/or GBL or a mixture of these twocomponents deriving from the culture using a heterogeneous orhomogeneous hydrogenation catalyst together with hydrogen, or ahydride-based reducing agent used stoichiometrically or catalytically,to produce tetrahydrofuran. Other procedures well know in the art areequally applicable for the above chemical reaction and include, forexample, U.S. Pat. No. 6,686,310, which describes high surface areasol-gel route prepared hydrogenation catalysts. Processes for thereduction of maleic acid to tetrahydrofuran (THF) and 1,4-butanediol(BDO) and for the reduction of gamma butyrolactone to tetrahydrofuranand 1,4-butanediol also are described.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described further below in the Examples, particularlyuseful yields of the biosynthetic products of the invention can beobtained under anaerobic or substantially anaerobic culture conditions.

Suitable purification and/or assays to test for the production of 4-HB,4-HBal, 4-HBCoA, BDO or putrescine can be performed using well knownmethods. Suitable replicates such as triplicate cultures can be grownfor each engineered strain to be tested. For example, product andbyproduct formation in the engineered production host can be monitored.The final product and intermediates, and other organic compounds, can beanalyzed by methods such as HPLC (High Performance LiquidChromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS(Liquid Chromatography-Mass Spectroscopy) or other suitable analyticalmethods using routine procedures well known in the art. The release ofproduct in the fermentation broth can also be tested with the culturesupernatant. Byproducts and residual glucose can be quantified by HPLCusing, for example, a refractive index detector for glucose andalcohols, and a UV detector for organic acids (Lin et al., Biotechnol.Bioeng. 90:775-779 (2005)), or other suitable assay and detectionmethods well known in the art. The individual enzyme or proteinactivities from the exogenous DNA sequences can also be assayed usingmethods well known in the art.

The 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine product can be separatedfrom other components in the culture using a variety of methods wellknown in the art. Such separation methods include, for example,extraction procedures as well as methods that include continuousliquid-liquid extraction, pervaporation, membrane filtration, membraneseparation, reverse osmosis, electrodialysis, distillation,crystallization, centrifugation, extractive filtration, ion exchangechromatography, size exclusion chromatography, adsorptionchromatography, and ultrafiltration. All of the above methods are wellknown in the art.

The invention further provides a method of manufacturing 4-HB. Themethod includes fermenting a non-naturally occurring microbial organismhaving a 4-hydroxybutanoic acid (4-HB) biosynthetic pathway comprisingat least one exogenous nucleic acid encoding 4-hydroxybutanoatedehydrogenase, CoA-independent succinic semialdehyde dehydrogenase,succinyl-CoA synthetase, CoA-dependent succinic semialdehydedehydrogenase, glutamate: succinic semialdehyde transaminase,α-ketoglutarate decarboxylase, or glutamate decarboxylase undersubstantially anaerobic conditions for a sufficient period of time toproduce monomeric 4-hydroxybutanoic acid (4-HB), the process comprisingfed-batch fermentation and batch separation; fed-batch fermentation andcontinuous separation, or continuous fermentation and continuousseparation.

The culture and chemical hydrogenations described above also can bescaled up and grown continuously for manufacturing of 4-HB, 4-HBal,4-HBCoA, GBL, BDO and/or THF or putrescine. Exemplary growth proceduresinclude, for example, fed-batch fermentation and batch separation;fed-batch fermentation and continuous separation, or continuousfermentation and continuous separation. All of these processes are wellknown in the art. Employing the 4-HB producers allows for simultaneous4-HB biosynthesis and chemical conversion to GBL, BDO and/or THF byemploying the above hydrogenation procedures simultaneous withcontinuous cultures methods such as fermentation. Other hydrogenationprocedures also are well known in the art and can be equally applied tothe methods of the invention.

Fermentation procedures are particularly useful for the biosyntheticproduction of commercial quantities of 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine. Generally, and as with non-continuous culture procedures,the continuous and/or near-continuous production of 4-HB, 4-HBal,4-HBCoA, BDO or putrescine will include culturing a non-naturallyoccurring 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine producing organism ofthe invention in sufficient nutrients and medium to sustain and/ornearly sustain growth in an exponential phase. Continuous culture undersuch conditions can be include, for example, 1 day, 2, 3, 4, 5, 6 or 7days or more. Additionally, continuous culture can include 1 week, 2, 3,4 or 5 or more weeks and up to several months. Alternatively, organismsof the invention can be cultured for hours, if suitable for a particularapplication. It is to be understood that the continuous and/ornear-continuous culture conditions also can include all time intervalsin between these exemplary periods. It is further understood that thetime of culturing the microbial organism of the invention is for asufficient period of time to produce a sufficient amount of product fora desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentationfor the biosynthetic production of 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine or other 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine derivedproducts, including intermediates, of the invention can be utilized in,for example, fed-batch fermentation and batch separation; fed-batchfermentation and continuous separation, or continuous fermentation andcontinuous separation. Examples of batch and continuous fermentationprocedures well known in the art are exemplified further below in theExamples.

In addition to the above fermentation procedures using the 4-HB, 4-HBal,4-HBCoA, BDO or putrescine producers of the invention for continuousproduction of substantial quantities of 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine, including monomeric 4-HB, respectively, the 4-HB producersalso can be, for example, simultaneously subjected to chemical synthesisprocedures to convert the product to other compounds or the product asdescribed previously for the chemical conversion of monomeric 4-HB to,for example, GBL, BDO and/or THF. The BDO producers can similarly be,for example, simultaneously subjected to chemical synthesis proceduresas described previously for the chemical conversion of BDO to, forexample, THF, GBL, pyrrolidones and/or other BDO family compounds. Inaddition, the products of the 4-HB, 4-HBal, 4-HBCoA, BDO or putrescineproducers can be separated from the fermentation culture andsequentially subjected to chemical or enzymatic conversion to convertthe product to other compounds, if desired, as disclosed herein.

Briefly, hydrogenation of GBL in the fermentation broth can be performedas described by Frost et al., Biotechnology Progress 18: 201-211 (2002).Another procedure for hydrogenation during fermentation include, forexample, the methods described in, for example, U.S. Pat. No. 5,478,952.This method is further exemplified in the Examples below.

Therefore, the invention additionally provides a method of manufacturingγ-butyrolactone (GBL), tetrahydrofuran (THF) or 1,4-butanediol (BDO).The method includes fermenting a non-naturally occurring microbialorganism having 4-hydroxybutanoic acid (4-HB) and/or 1,4-butanediol(BDO) biosynthetic pathways, the pathways comprise at least oneexogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase,CoA-independent succinic semialdehyde dehydrogenase, succinyl-CoAsynthetase, CoA-dependent succinic semialdehyde dehydrogenase,4-hydroxybutyrate:CoA transferase, glutamate: succinic semialdehydetransaminase, α-ketoglutarate decarboxylase, glutamate decarboxylase,4-hydroxybutanoate kinase, phosphotransbutyrylase, CoA-independent1,4-butanediol semialdehyde dehydrogenase, CoA-dependent 1,4-butanediolsemialdehyde dehydrogenase, CoA-independent 1,4-butanediol alcoholdehydrogenase or CoA-dependent 1,4-butanediol alcohol dehydrogenase,under substantially anaerobic conditions for a sufficient period of timeto produce 1,4-butanediol (BDO), GBL or THF, the fermenting comprisingfed-batch fermentation and batch separation; fed-batch fermentation andcontinuous separation, or continuous fermentation and continuousseparation.

In addition to the biosynthesis of 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine and other products of the invention as described herein, thenon-naturally occurring microbial organisms and methods of the inventionalso can be utilized in various combinations with each other and/or withother microbial organisms and methods well known in the art to achieveproduct biosynthesis by other routes. For example, one alternative toproduce BDO other than use of the 4-HB producers and chemical steps orother than use of the BDO producer directly is through addition ofanother microbial organism capable of converting 4-HB or a 4-HB productexemplified herein to BDO.

One such procedure includes, for example, the fermentation of a 4-HBproducing microbial organism of the invention to produce 4-HB, asdescribed above and below. The 4-HB can then be used as a substrate fora second microbial organism that converts 4-HB to, for example, BDO, GBLand/or THF. The 4-HB can be added directly to another culture of thesecond organism or the original culture of 4-HB producers can bedepleted of these microbial organisms by, for example, cell separation,and then subsequent addition of the second organism to the fermentationbroth can utilized to produce the final product without intermediatepurification steps. One exemplary second organism having the capacity tobiochemically utilize 4-HB as a substrate for conversion to BDO, forexample, is Clostridium acetobutylicum (see, for example, Jewell et al.,Current Microbiology, 13:215-19 (1986)).

Thus, such a procedure includes, for example, the fermentation of amicrobial organism that produces a 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine pathway intermediate. The 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine pathway intermediate can then be used as a substrate for asecond microbial organism that converts the 4-HB, 4-HBal, 4-HBCoA, BDOor putrescine pathway intermediate to 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine. The 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine pathwayintermediate can be added directly to another culture of the secondorganism or the original culture of the 4-HB, 4-HBal, 4-HBCoA BDO orputrescine pathway intermediate producers can be depleted of thesemicrobial organisms by, for example, cell separation, and thensubsequent addition of the second organism to the fermentation broth canbe utilized to produce the final product without intermediatepurification steps.

In other embodiments, the non-naturally occurring microbial organismsand methods of the invention can be assembled in a wide variety ofsubpathways to achieve biosynthesis of, for example, 4-HB and/or BDO asdescribed. In these embodiments, biosynthetic pathways for a desiredproduct of the invention can be segregated into different microbialorganisms and the different microbial organisms can be co-cultured toproduce the final product. In such a biosynthetic scheme, the product ofone microbial organism is the substrate for a second microbial organismuntil the final product is synthesized. For example, the biosynthesis ofBDO can be accomplished as described previously by constructing amicrobial organism that contains biosynthetic pathways for conversion ofone pathway intermediate to another pathway intermediate or the product,for example, a substrate such as endogenous succinate through 4-HB tothe final product BDO. Alternatively, BDO also can be biosyntheticallyproduced from microbial organisms through co-culture or co-fermentationusing two organisms in the same vessel. A first microbial organism beinga 4-HB producer with genes to produce 4-HB from succinic acid, and asecond microbial organism being a BDO producer with genes to convert4-HB to BDO. For example, the biosynthesis of 4-HB, 4-HBal, 4-HBCoA, BDOor putrescine can be accomplished by constructing a microbial organismthat contains biosynthetic pathways for conversion of one pathwayintermediate to another pathway intermediate or the product.Alternatively, 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine also can bebiosynthetically produced from microbial organisms through co-culture orco-fermentation using two organisms in the same vessel, where the firstmicrobial organism produces a 4-HB, 4-HBal, 4-HBCoA, BDO or putrescineintermediate and the second microbial organism converts the intermediateto 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine.

Given the teachings and guidance provided herein, those skilled in theart will understand that a wide variety of combinations and permutationsexist for the non-naturally occurring microbial organisms and methods ofthe invention together with other microbial organisms, with theco-culture of other non-naturally occurring microbial organisms havingsubpathways and with combinations of other chemical and/or biochemicalprocedures well known in the art to produce 4-HB, BDO, GBL, THF andputrescine products of the invention.

Similarly, it is understood by those skilled in the art that a hostorganism can be selected based on desired characteristics forintroduction of one or more gene disruptions to increase production of4-HB, 4-HBal, 4-HBCoA, BDO or putrescine. Thus, it is understood that,if a genetic modification is to be introduced into a host organism todisrupt a gene, any homologs, orthologs or paralogs that catalyzesimilar, yet non-identical metabolic reactions can similarly bedisrupted to ensure that a desired metabolic reaction is sufficientlydisrupted. Because certain differences exist among metabolic networksbetween different organisms, those skilled in the art will understandthat the actual genes disrupted in a given organism may differ betweenorganisms. However, given the teachings and guidance provided herein,those skilled in the art also will understand that the methods of theinvention can be applied to any suitable host microorganism to identifythe cognate metabolic alterations needed to construct an organism in aspecies of interest that will increase 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine biosynthesis. In a particular embodiment, the increasedproduction couples biosynthesis of 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine to growth of the organism, and can obligatorily coupleproduction of 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine to growth of theorganism if desired and as disclosed herein.

It is understood that, in methods of the invention, any of the one ormore exogenous nucleic acids can be introduced into a microbial organismto produce a non-naturally occurring microbial organism of theinvention. The nucleic acids can be introduced so as to confer, forexample, a 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine biosynthetic pathwayonto the microbial organism. Alternatively, encoding nucleic acids canbe introduced to produce an intermediate microbial organism having thebiosynthetic capability to catalyze some of the required reactions toconfer 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine biosynthetic capability.For example, a non-naturally occurring microbial organism having a 4-HB,4-HBal, 4-HBCoA, BDO or putrescine biosynthetic pathway can comprise atleast two exogenous nucleic acids encoding desired enzymes or proteins,such as the combination of enzymes as disclosed herein (see Examples andFIGS. 1, 8-13, 58, 62 and 63), and the like. Thus, it is understood thatany combination of two or more enzymes or proteins of a biosyntheticpathway can be included in a non-naturally occurring microbial organismof the invention. Similarly, it is understood that any combination ofthree or more enzymes or proteins of a biosynthetic pathway can beincluded in a non-naturally occurring microbial organism of theinvention, for example, and so forth, as desired and disclosed herein,so long as the combination of enzymes and/or proteins of the desiredbiosynthetic pathway results in production of the corresponding desiredproduct. Similarly, any combination of four or more enzymes or proteinsof a biosynthetic pathway as disclosed herein can be included in anon-naturally occurring microbial organism of the invention, as desired,so long as the combination of enzymes and/or proteins of the desiredbiosynthetic pathway results in production of the corresponding desiredproduct.

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. patent publications US 2002/0012939, US 2003/0224363,US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 andUS 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allowsreliable predictions of the effects on cell growth of shifting themetabolism towards more efficient production of 4-HB, 4-HBal, 4-HBCoA,BDO or putrescine.

One computational method for identifying and designing metabolicalterations favoring biosynthesis of a desired product is the OptKnockcomputational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657(2003)). OptKnock is a metabolic modeling and simulation program thatsuggests gene deletion or disruption strategies that result ingenetically stable microorganisms which overproduce the target product.Specifically, the framework examines the complete metabolic and/orbiochemical network of a microorganism in order to suggest geneticmanipulations that force the desired biochemical to become an obligatorybyproduct of cell growth. By coupling biochemical production with cellgrowth through strategically placed gene deletions or other functionalgene disruption, the growth selection pressures imposed on theengineered strains after long periods of time in a bioreactor lead toimprovements in performance as a result of the compulsory growth-coupledbiochemical production. Lastly, when gene deletions are constructedthere is a negligible possibility of the designed strains reverting totheir wild-type states because the genes selected by OptKnock are to becompletely removed from the genome. Therefore, this computationalmethodology can be used to either identify alternative pathways thatlead to biosynthesis of a desired product or used in connection with thenon-naturally occurring microbial organisms for further optimization ofbiosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or deletions. OptKnockcomputational framework allows the construction of model formulationsthat allow an effective query of the performance limits of metabolicnetworks and provides methods for solving the resulting mixed-integerlinear programming problems. The metabolic modeling and simulationmethods referred to herein as OptKnock are described in, for example,U.S. publication 2002/0168654, filed Jan. 10, 2002, in InternationalPatent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. publication2009/0047719, filed Aug. 10, 2007.

Another computational method for identifying and designing metabolicalterations favoring biosynthetic production of a product is a metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. publication2003/0233218, filed Jun. 14, 2002, and in International PatentApplication No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is acomputational system that can be used to produce a network model insilico and to simulate the flux of mass, energy or charge through thechemical reactions of a biological system to define a solution spacethat contains any and all possible functionalities of the chemicalreactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realitiesbecause biological systems are flexible and can reach the same result inmany different ways. Biological systems are designed throughevolutionary mechanisms that have been restricted by fundamentalconstraints that all living systems must face. Therefore,constraints-based modeling strategy embraces these general realities.Further, the ability to continuously impose further restrictions on anetwork model via the tightening of constraints results in a reductionin the size of the solution space, thereby enhancing the precision withwhich physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in theart will be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of adesired compound in host microbial organisms. Such metabolic modelingand simulation methods include, for example, the computational systemsexemplified above as SimPheny® and OptKnock. For illustration of theinvention, some methods are described herein with reference to theOptKnock computation framework for modeling and simulation. Thoseskilled in the art will know how to apply the identification, design andimplementation of the metabolic alterations using OptKnock to any ofsuch other metabolic modeling and simulation computational frameworksand methods well known in the art.

The methods described above will provide one set of metabolic reactionsto disrupt. Elimination of each reaction within the set or metabolicmodification can result in a desired product as an obligatory productduring the growth phase of the organism. Because the reactions areknown, a solution to the bilevel OptKnock problem also will provide theassociated gene or genes encoding one or more enzymes that catalyze eachreaction within the set of reactions. Identification of a set ofreactions and their corresponding genes encoding the enzymesparticipating in each reaction is generally an automated process,accomplished through correlation of the reactions with a reactiondatabase having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in orderto achieve production of a desired product are implemented in the targetcell or organism by functional disruption of at least one gene encodingeach metabolic reaction within the set. One particularly useful means toachieve functional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the coupling of a product are desiredor when genetic reversion is less likely to occur.

To identify additional productive solutions to the above describedbilevel OptKnock problem which lead to further sets of reactions todisrupt or metabolic modifications that can result in the biosynthesis,including growth-coupled biosynthesis of a desired product, anoptimization method, termed integer cuts, can be implemented. Thismethod proceeds by iteratively solving the OptKnock problem exemplifiedabove with the incorporation of an additional constraint referred to asan integer cut at each iteration. Integer cut constraints effectivelyprevent the solution procedure from choosing the exact same set ofreactions identified in any previous iteration that obligatorily couplesproduct biosynthesis to growth. For example, if a previously identifiedgrowth-coupled metabolic modification specifies reactions 1, 2, and 3for disruption, then the following constraint prevents the samereactions from being simultaneously considered in subsequent solutions.The integer cut method is well known in the art and can be founddescribed in, for example, Burgard et al., Biotechnol. Prog. 17:791-797(2001). As with all methods described herein with reference to their usein combination with the OptKnock computational framework for metabolicmodeling and simulation, the integer cut method of reducing redundancyin iterative computational analysis also can be applied with othercomputational frameworks well known in the art including, for example,SimPheny®.

The methods exemplified herein allow the construction of cells andorganisms that biosynthetically produce a desired product, including theobligatory coupling of production of a target biochemical product togrowth of the cell or organism engineered to harbor the identifiedgenetic alterations. Therefore, the computational methods describedherein allow the identification and implementation of metabolicmodifications that are identified by an in silico method selected fromOptKnock or SimPheny®. The set of metabolic modifications can include,for example, addition of one or more biosynthetic pathway enzymes and/orfunctional disruption of one or more metabolic reactions including, forexample, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on thepremise that mutant microbial networks can be evolved towards theircomputationally predicted maximum-growth phenotypes when subjected tolong periods of growth selection. In other words, the approach leveragesan organism's ability to self-optimize under selective pressures. TheOptKnock framework allows for the exhaustive enumeration of genedeletion combinations that force a coupling between biochemicalproduction and cell growth based on network stoichiometry. Theidentification of optimal gene/reaction knockouts requires the solutionof a bilevel optimization problem that chooses the set of activereactions such that an optimal growth solution for the resulting networkoverproduces the biochemical of interest (Burgard et al., Biotechnol.Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employedto identify essential genes for metabolic pathways as exemplifiedpreviously and described in, for example, U.S. patent publications US2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No.7,127,379. As disclosed herein, the OptKnock mathematical framework canbe applied to pinpoint gene deletions leading to the growth-coupledproduction of a desired product. Further, the solution of the bilevelOptKnock problem provides only one set of deletions. To enumerate allmeaningful solutions, that is, all sets of knockouts leading togrowth-coupled production formation, an optimization technique, termedinteger cuts, can be implemented. This entails iteratively solving theOptKnock problem with the incorporation of an additional constraintreferred to as an integer cut at each iteration, as discussed above.

The methods exemplified above and further illustrated in the Examplesbelow allow the construction of cells and organisms thatbiosynthetically produce, including obligatory couple production of atarget biochemical product to growth of the cell or organism engineeredto harbor the identified genetic alterations. In this regard, metabolicalterations have been identified that result in the biosynthesis of 4-HBand 1,4-butanediol. Microorganism strains constructed with theidentified metabolic alterations produce elevated levels of 4-HB,4-HBal, 4-HBCoA, BDO or putrescine compared to unmodified microbialorganisms. These strains can be beneficially used for the commercialproduction of 4-HB, BDO, THF, GBL, 4-HBal, 4-HBCoA or putrescine, forexample, in continuous fermentation process without being subjected tothe negative selective pressures.

Therefore, the computational methods described herein allow theidentification and implementation of metabolic modifications that areidentified by an in silico method selected from OptKnock or SimPheny®.The set of metabolic modifications can include, for example, addition ofone or more biosynthetic pathway enzymes and/.or functional disruptionof one or more metabolic reactions including, for example, disruption bygene deletion.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoincluded within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

Any of the non-naturally occurring microbial organisms described hereincan be cultured to produce and/or secrete the biosynthetic products ofthe invention. For example, the 4-HB, 4-HBal, 4-HBCoA, BDO or putrescineproducers can be cultured for the biosynthetic production of 4-HB,4-HBal, 4-HBCoA, BDO or putrescine.

For the production of 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine, therecombinant strains are cultured in a medium with carbon source andother essential nutrients. It is highly desirable to maintain anaerobicconditions in the fermenter to reduce the cost of the overall process.Such conditions can be obtained, for example, by first sparging themedium with nitrogen and then sealing the flasks with a septum andcrimp-cap. For strains where growth is not observed anaerobically,microaerobic conditions can be applied by perforating the septum with asmall hole for limited aeration. Exemplary anaerobic conditions havebeen described previously and are well-known in the art. Exemplaryaerobic and anaerobic conditions are described, for example, in U.S.publication 2009/0047719, filed Aug. 10, 2007. Fermentations can beperformed in a batch, fed-batch or continuous manner, as disclosedherein.

If desired, the pH of the medium can be maintained at a desired pH, inparticular neutral pH, such as a pH of around 7 by addition of a base,such as NaOH or other bases, or acid, as needed to maintain the culturemedium at a desirable pH. The growth rate can be determined by measuringoptical density using a spectrophotometer (600 nm), and the glucoseuptake rate by monitoring carbon source depletion over time.

In addition to renewable feedstocks such as those exemplified above, the4-HB, 4-HBal, 4-HBCoA, BDO or putrescine producing microbial organismsof the invention also can be modified for growth on syngas as its sourceof carbon. In this specific embodiment, one or more proteins or enzymesare expressed in the 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine producingorganisms to provide a metabolic pathway for utilization of syngas orother gaseous carbon source.

Synthesis gas, also known as syngas or producer gas, is the majorproduct of gasification of coal and of carbonaceous materials such asbiomass materials, including agricultural crops and residues. Syngas isa mixture primarily of H₂ and CO and can be obtained from thegasification of any organic feedstock, including but not limited tocoal, coal oil, natural gas, biomass, and waste organic matter.Gasification is generally carried out under a high fuel to oxygen ratio.Although largely H₂ and CO, syngas can also include CO₂ and other gasesin smaller quantities. Thus, synthesis gas provides a cost effectivesource of gaseous carbon such as CO and, additionally, CO₂.

The Wood-Ljungdahl pathway catalyzes the conversion of CO and H₂ toacetyl-CoA and other products such as acetate. Organisms capable ofutilizing CO and syngas also generally have the capability of utilizingCO₂ and CO₂/H₂ mixtures through the same basic set of enzymes andtransformations encompassed by the Wood-Ljungdahl pathway. H₂-dependentconversion of CO₂ to acetate by microorganisms was recognized longbefore it was revealed that CO also could be used by the same organismsand that the same pathways were involved. Many acetogens have been shownto grow in the presence of CO₂ and produce compounds such as acetate aslong as hydrogen is present to supply the necessary reducing equivalents(see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, NewYork, (1994)). This can be summarized by the following equation:2CO₂+4H₂ +nADP+nPi→CH₃COOH+2H₂O+nATPHence, non-naturally occurring microorganisms possessing theWood-Ljungdahl pathway can utilize CO₂ and H₂ mixtures as well for theproduction of acetyl-CoA and other desired products.

The Wood-Ljungdahl pathway is well known in the art and consists of 12reactions which can be separated into two branches: (1) methyl branchand (2) carbonyl branch. The methyl branch converts syngas tomethyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branchconverts methyl-THF to acetyl-CoA. The reactions in the methyl branchare catalyzed in order by the following enzymes or proteins: ferredoxinoxidoreductase, formate dehydrogenase, formyltetrahydrofolatesynthetase, methenyltetrahydrofolate cyclodehydratase,methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolatereductase. The reactions in the carbonyl branch are catalyzed in orderby the following enzymes or proteins: methyltetrahydrofolate:corrinoidprotein methyltransferase (for example, AcsE), corrinoid iron-sulfurprotein, nickel-protein assembly protein (for example, AcsF),ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase andnickel-protein assembly protein (for example, CooC). Following theteachings and guidance provided herein for introducing a sufficientnumber of encoding nucleic acids to generate a 4-HB, 4-HBal, 4-HBCoA,BDO or putrescine pathway, those skilled in the art will understand thatthe same engineering design also can be performed with respect tointroducing at least the nucleic acids encoding the Wood-Ljungdahlenzymes or proteins absent in the host organism. Therefore, introductionof one or more encoding nucleic acids into the microbial organisms ofthe invention such that the modified organism contains the completeWood-Ljungdahl pathway will confer syngas utilization ability.

Additionally, the reductive (reverse) tricarboxylic acid cycle is and/orhydrogenase activities can also be used for the conversion of CO, CO₂and/or H₂ to acetyl-CoA and other products such as acetate. Organismscapable of fixing carbon via the reductive TCA pathway can utilize oneor more of the following enzymes: ATP citrate-lyase, citrate lyase,aconitase, isocitrate dehydrogenase, alpha-ketoglutarate:ferredoxinoxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase,fumarate reductase, fumarase, malate dehydrogenase, NAD(P)H:ferredoxinoxidoreductase, carbon monoxide dehydrogenase, and hydrogenase.Specifically, the reducing equivalents extracted from CO and/or H2 bycarbon monoxide dehydrogenase and hydrogenase are utilized to fix CO2via the reductive TCA cycle into acetyl-CoA or acetate. Acetate can beconverted to acetyl-CoA by enzymes such as acetyl-CoA transferase,acetate kinase/phosphotransacetylase, and acetyl-CoA synthetase.Acetyl-CoA can be converted to the 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine precursors, glyceraldehyde-3-phosphate, phosphoenolpyruvate,and pyruvate, by pyruvate:ferredoxin oxidoreductase and the enzymes ofgluconeogenesis. Following the teachings and guidance provided hereinfor introducing a sufficient number of encoding nucleic acids togenerate a 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine pathway, thoseskilled in the art will understand that the same engineering design alsocan be performed with respect to introducing at least the nucleic acidsencoding the reductive TCA pathway enzymes or proteins absent in thehost organism. Therefore, introduction of one or more encoding nucleicacids into the microbial organisms of the invention such that themodified organism contains the complete reductive TCA pathway willconfer syngas utilization ability.

Accordingly, given the teachings and guidance provided herein, thoseskilled in the art will understand that a non-naturally occurringmicrobial organism can be produced that secretes the biosynthesizedcompounds of the invention when grown on a carbon source such as acarbohydrate. Such compounds include, for example, 4-HB, 4-HBal,4-HBCoA, BDO or putrescine and any of the intermediate metabolites inthe 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine pathway. All that isrequired is to engineer in one or more of the required enzyme or proteinactivities to achieve biosynthesis of the desired compound orintermediate including, for example, inclusion of some or all of the4-HB, 4-HBal, 4-HBCoA, BDO or putrescine biosynthetic pathways.Accordingly, the invention provides a non-naturally occurring microbialorganism that produces and/or secretes 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine when grown on a carbohydrate or other carbon source andproduces and/or secretes any of the intermediate metabolites shown inthe 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine pathway when grown on acarbohydrate or other carbon source. The 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine producing microbial organisms of the invention can initiatesynthesis from an intermediate in a 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine pathway, as disclosed herein.

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. patent publications US 2002/0012939, US 2003/0224363,US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 andUS 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allowsreliable predictions of the effects on cell growth of shifting themetabolism towards more efficient production of 4-HB, 4-HBal, 4-HBCoA,BDO or putrescine.

One computational method for identifying and designing metabolicalterations favoring biosynthesis of a desired product is the OptKnockcomputational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657(2003)). OptKnock is a metabolic modeling and simulation program thatsuggests gene deletion strategies that result in genetically stablemicroorganisms which overproduce the target product. Specifically, theframework examines the complete metabolic and/or biochemical network ofa microorganism in order to suggest genetic manipulations that force thedesired biochemical to become an obligatory byproduct of cell growth. Bycoupling biochemical production with cell growth through strategicallyplaced gene deletions or other functional gene disruption, the growthselection pressures imposed on the engineered strains after long periodsof time in a bioreactor lead to improvements in performance as a resultof the compulsory growth-coupled biochemical production. Lastly, whengene deletions are constructed there is a negligible possibility of thedesigned strains reverting to their wild-type states because the genesselected by OptKnock are to be completely removed from the genome.Therefore, this computational methodology can be used to either identifyalternative pathways that lead to biosynthesis of a desired product orused in connection with the non-naturally occurring microbial organismsfor further optimization of biosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or deletions. OptKnockcomputational framework allows the construction of model formulationsthat allow an effective query of the performance limits of metabolicnetworks and provides methods for solving the resulting mixed-integerlinear programming problems. The metabolic modeling and simulationmethods referred to herein as OptKnock are described in, for example,U.S. publication 2002/0168654, filed Jan. 10, 2002, in InternationalPatent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. publication2009/0047719, filed Aug. 10, 2007.

Another computational method for identifying and designing metabolicalterations favoring biosynthetic production of a product is a metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. publication2003/0233218, filed Jun. 14, 2002, and in International PatentApplication No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is acomputational system that can be used to produce a network model insilico and to simulate the flux of mass, energy or charge through thechemical reactions of a biological system to define a solution spacethat contains any and all possible functionalities of the chemicalreactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realitiesbecause biological systems are flexible and can reach the same result inmany different ways. Biological systems are designed throughevolutionary mechanisms that have been restricted by fundamentalconstraints that all living systems must face. Therefore,constraints-based modeling strategy embraces these general realities.Further, the ability to continuously impose further restrictions on anetwork model via the tightening of constraints results in a reductionin the size of the solution space, thereby enhancing the precision withwhich physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in theart will be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of adesired compound in host microbial organisms. Such metabolic modelingand simulation methods include, for example, the computational systemsexemplified above as SimPheny® and OptKnock. For illustration of theinvention, some methods are described herein with reference to theOptKnock computation framework for modeling and simulation. Thoseskilled in the art will know how to apply the identification, design andimplementation of the metabolic alterations using OptKnock to any ofsuch other metabolic modeling and simulation computational frameworksand methods well known in the art.

The methods described above will provide one set of metabolic reactionsto disrupt. Elimination of each reaction within the set or metabolicmodification can result in a desired product as an obligatory productduring the growth phase of the organism. Because the reactions areknown, a solution to the bilevel OptKnock problem also will provide theassociated gene or genes encoding one or more enzymes that catalyze eachreaction within the set of reactions. Identification of a set ofreactions and their corresponding genes encoding the enzymesparticipating in each reaction is generally an automated process,accomplished through correlation of the reactions with a reactiondatabase having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in orderto achieve production of a desired product are implemented in the targetcell or organism by functional disruption of at least one gene encodingeach metabolic reaction within the set. One particularly useful means toachieve functional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the coupling of a product are desiredor when genetic reversion is less likely to occur.

To identify additional productive solutions to the above describedbilevel OptKnock problem which lead to further sets of reactions todisrupt or metabolic modifications that can result in the biosynthesis,including growth-coupled biosynthesis of a desired product, anoptimization method, termed integer cuts, can be implemented. Thismethod proceeds by iteratively solving the OptKnock problem exemplifiedabove with the incorporation of an additional constraint referred to asan integer cut at each iteration. Integer cut constraints effectivelyprevent the solution procedure from choosing the exact same set ofreactions identified in any previous iteration that obligatorily couplesproduct biosynthesis to growth. For example, if a previously identifiedgrowth-coupled metabolic modification specifies reactions 1, 2, and 3for disruption, then the following constraint prevents the samereactions from being simultaneously considered in subsequent solutions.The integer cut method is well known in the art and can be founddescribed in, for example, Burgard et al., Biotechnol. Prog. 17:791-797(2001). As with all methods described herein with reference to their usein combination with the OptKnock computational framework for metabolicmodeling and simulation, the integer cut method of reducing redundancyin iterative computational analysis also can be applied with othercomputational frameworks well known in the art including, for example,SimPheny®.

The methods exemplified herein allow the construction of cells andorganisms that biosynthetically produce a desired product, including theobligatory coupling of production of a target biochemical product togrowth of the cell or organism engineered to harbor the identifiedgenetic alterations. Therefore, the computational methods describedherein allow the identification and implementation of metabolicmodifications that are identified by an in silico method selected fromOptKnock or SimPheny®. The set of metabolic modifications can include,for example, addition of one or more biosynthetic pathway enzymes and/orfunctional disruption of one or more metabolic reactions including, forexample, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on thepremise that mutant microbial networks can be evolved towards theircomputationally predicted maximum-growth phenotypes when subjected tolong periods of growth selection. In other words, the approach leveragesan organism's ability to self-optimize under selective pressures. TheOptKnock framework allows for the exhaustive enumeration of genedeletion combinations that force a coupling between biochemicalproduction and cell growth based on network stoichiometry. Theidentification of optimal gene/reaction knockouts requires the solutionof a bilevel optimization problem that chooses the set of activereactions such that an optimal growth solution for the resulting networkoverproduces the biochemical of interest (Burgard et al., Biotechnol.Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employedto identify essential genes for metabolic pathways as exemplifiedpreviously and described in, for example, U.S. patent publications US2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No.7,127,379. As disclosed herein, the OptKnock mathematical framework canbe applied to pinpoint gene deletions leading to the growth-coupledproduction of a desired product. Further, the solution of the bilevelOptKnock problem provides only one set of deletions. To enumerate allmeaningful solutions, that is, all sets of knockouts leading togrowth-coupled production formation, an optimization technique, termedinteger cuts, can be implemented. This entails iteratively solving theOptKnock problem with the incorporation of an additional constraintreferred to as an integer cut at each iteration, as discussed above.

Employing the methods exemplified above and herein, the methods of theinvention allow the construction of cells and organisms that increaseproduction of a desired product, for example, by coupling the productionof a desired product to growth of the cell or organism engineered toharbor the identified genetic alterations. As disclosed herein,metabolic alterations have been identified that couple the production of4-HB, 4-HBal, 4-HBCoA, BDO or putrescine to growth of the organism.Microbial organism strains constructed with the identified metabolicalterations produce elevated levels, relative to the absence of themetabolic alterations, of 4-HB, 4-HBal, 4-HBCoA, BDO or putrescineduring the exponential growth phase. These strains can be beneficiallyused for the commercial production of 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine in continuous fermentation process without being subjected tothe negative selective pressures described previously. Althoughexemplified herein as metabolic alterations, in particular one or moregene disruptions, that confer growth coupled production of 4-HB, 4-HBal,4-HBCoA, BDO or putrescine, it is understood that any gene disruptionthat increases the production of 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine can be introduced into a host microbial organism, as desired.

Therefore, the methods of the invention provide a set of metabolicmodifications that are identified by an in silico method such asOptKnock. The set of metabolic modifications can include functionaldisruption of one or more metabolic reactions including, for example,disruption by gene deletion. For 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine production, metabolic modifications can be selected from theset of metabolic modifications described herein, including the Examples.

Also provided is a method of producing a non-naturally occurringmicrobial organisms having stable growth-coupled production of 4-HB,4-HBal, 4-HBCoA, BDO or putrescine. The method can include identifyingin silico a set of metabolic modifications that increase production of4-HB, 4-HBal, 4-HBCoA, BDO or putrescine, for example, increaseproduction during exponential growth; genetically modifying an organismto contain the set of metabolic modifications that increase productionof 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine, and culturing thegenetically modified organism. If desired, culturing can includeadaptively evolving the genetically modified organism under conditionsrequiring production of 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine. Themethods of the invention are applicable to bacterium, yeast and fungusas well as a variety of other cells and microorganism, as disclosedherein.

Thus, the invention provides a non-naturally occurring microbialorganism comprising one or more gene disruptions that confer increasedproduction of 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine. In oneembodiment, the one or more gene disruptions confer growth-coupledproduction of 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine, and can, forexample, confer stable growth-coupled production of 4-HB, 4-HBal,4-HBCoA, BDO or putrescine. In another embodiment, the one or more genedisruptions can confer obligatory coupling of 4-HB, 4-HBal, 4-HBCoA, BDOor putrescine production to growth of the microbial organism. Such oneor more gene disruptions reduce the activity of the respective one ormore encoded enzymes.

The non-naturally occurring microbial organism can have one or more genedisruptions included in a metabolic modification as described herein. Asdisclosed herein, the one or more gene disruptions can be a deletion.Such non-naturally occurring microbial organisms of the inventioninclude bacteria, yeast, fungus, or any of a variety of othermicroorganisms applicable to fermentation processes, as disclosedherein.

Thus, the invention provides a non-naturally occurring microbialorganism, comprising one or more gene disruptions, where the one or moregene disruptions occur in genes encoding proteins or enzymes where theone or more gene disruptions confer increased production of 4-HB,4-HBal, 4-HBCoA, BDO or putrescine in the organism. The production of4-HB, 4-HBal, 4-HBCoA, BDO or putrescine can be growth-coupled or notgrowth-coupled. In a particular embodiment, the production of 4-HB,4-HBal, 4-HBCoA, BDO or putrescine can be obligatorily coupled to growthof the organism, as disclosed herein.

The invention provides non naturally occurring microbial organismshaving genetic alterations such as gene disruptions that increaseproduction of 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine, for example,growth-coupled production of 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine.Product production can be, for example, obligatorily linked to theexponential growth phase of the microorganism by genetically alteringthe metabolic pathways of the cell, as disclosed herein. The geneticalterations can increase the production of the desired product or evenmake the desired product an obligatory product during the growth phase.Sets of metabolic alterations or transformations that result inincreased production and elevated levels of 4-HB, 4-HBal, 4-HBCoA, BDOor putrescine biosynthesis are described herein. Each alteration withina set corresponds to the requisite metabolic reaction that should befunctionally disrupted. Functional disruption of all reactions withineach set can result in the increased production of 4-HB, 4-HBal,4-HBCoA, BDO or putrescine by the engineered strain during the growthphase.

A number of metabolic modifications that include gene disruptions aredescribed herein. It is understood by those skilled in the art that oneor more of the metabolic modifications, including gene disruptions, canbe combined to increase 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine orotherwise improve characteristics of the microorganisms of theinvention. Each of these non-naturally occurring alterations can resultin increased production and an enhanced level of 4-HB, 4-HBal, 4-HBCoA,BDO or putrescine production, for example, during the exponential growthphase of the microbial organism, or otherwise improve the growth orproduction characteristics of the microorganisms producing 4-HB, 4-HBal,4-HBCoA, BDO or putrescine compared to a strain that does not containsuch metabolic alterations, under appropriate culture conditions.Appropriate conditions include, for example, those disclosed herein,including conditions such as particular carbon sources or reactantavailabilities and/or adaptive evolution.

Given the teachings and guidance provided herein, those skilled in theart will understand that to introduce a metabolic alteration such asdisruption of an enzymatic reaction, it is necessary to disrupt thecatalytic activity of the one or more enzymes involved in the reaction.Alternatively, a metabolic alteration can include disruption ofexpression of a regulatory protein or cofactor necessary for enzymeactivity or maximal activity. Disruption can occur by a variety ofmethods including, for example, deletion of an encoding gene orincorporation of a genetic alteration in one or more of the encodinggene sequences. The encoding genes targeted for disruption can be one,some, or all of the genes encoding enzymes involved in the catalyticactivity. For example, where a single enzyme is involved in a targetedcatalytic activity, disruption can occur by a genetic alteration thatreduces or eliminates the catalytic activity of the encoded geneproduct. Similarly, where the single enzyme is multimeric, includingheteromeric, disruption can occur by a genetic alteration that reducesor destroys the function of one or all subunits of the encoded geneproducts. Destruction of activity can be accomplished by loss of thebinding activity of one or more subunits required to form an activecomplex, by destruction of the catalytic subunit of the multimericcomplex or by both. Other functions of multimeric protein associationand activity also can be targeted in order to disrupt a metabolicreaction of the invention. Such other functions are well known to thoseskilled in the art. Similarly, a target enzyme activity can be reducedor eliminated by disrupting expression of a protein or enzyme thatmodifies and/or activates the target enzyme, for example, a moleculerequired to convert an apoenzyme to a holoenzyme. Further, some or allof the functions of a single polypeptide or multimeric complex can bedisrupted according to the invention in order to reduce or abolish thecatalytic activity of one or more enzymes involved in a reaction ormetabolic modification of the invention. Similarly, some or all ofenzymes involved in a reaction or metabolic modification of theinvention can be disrupted so long as the targeted reaction is reducedor eliminated.

Given the teachings and guidance provided herein, those skilled in theart also will understand that an enzymatic reaction can be disrupted byreducing or eliminating reactions encoded by a common gene and/or by oneor more orthologs of that gene exhibiting similar or substantially thesame activity. Reduction of both the common gene and all orthologs canlead to complete abolishment of any catalytic activity of a targetedreaction. However, disruption of either the common gene or one or moreorthologs can lead to a reduction in the catalytic activity of thetargeted reaction sufficient to promote coupling of growth to productbiosynthesis. Exemplified herein are both the common genes encodingcatalytic activities for a variety of metabolic modifications as well astheir orthologs. Those skilled in the art will understand thatdisruption of some or all of the genes encoding a enzyme of a targetedmetabolic reaction can be practiced in the methods of the invention andincorporated into the non-naturally occurring microbial organisms of theinvention in order to achieve the increased production of 4-HB, 4-HBal,4-HBCoA, BDO or putrescine or growth-coupled product production.

In some embodiments, microaerobic designs can be used based on thegrowth-coupled formation of the desired product. To examine this,production cones can be constructed for each strategy by firstmaximizing and, subsequently minimizing the product yields at differentrates of biomass formation feasible in the network. If the rightmostboundary of all possible phenotypes of the mutant network is a singlepoint, it implies that there is a unique optimum yield of the product atthe maximum biomass formation rate possible in the network. In othercases, the rightmost boundary of the feasible phenotypes is a verticalline, indicating that at the point of maximum biomass the network canmake any amount of the product in the calculated range, including thelowest amount at the bottommost point of the vertical line. Such designsare given a low priority.

The 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine production strategiesidentified by the methods disclosed herein such as the OptKnockframework are generally ranked on the basis of their (i) theoreticalyields, and optionally (ii) growth-coupled 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine formation characteristics.

Accordingly, the invention also provides a non-naturally occurringmicrobial organism having a set of metabolic modifications that increasethe yield of 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine, optionallycoupling 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine production to growthof the organism, or otherwise improve characteristics of themicroorganisms producing 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine, wherethe set of metabolic modifications includes disruption of one or moregenes, as described herein.

Each of the strains can be supplemented with additional deletions if itis determined that the strain designs do not sufficiently increase theproduction of 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine and/or couple theformation of the product with biomass formation. Alternatively, someother enzymes not known to possess significant activity under the growthconditions can become active due to adaptive evolution or randommutagenesis. Such activities can also be knocked out. However, the listof gene deletion sets disclosed herein allows the construction ofstrains exhibiting high-yield production of 4-HB, 4-HBal, 4-HBCoA, BDOor putrescine, including growth-coupled production of 4-HB, 4-HBal,4-HBCoA, BDO or putrescine.

4-HB, 4-HBal, 4-HBCoA, BDO or putrescine can be harvested or isolated atany time point during the culturing of the microbial organism, forexample, in a continuous and/or near-continuous culture period, asdisclosed herein. Generally, the longer the microorganisms aremaintained in a continuous and/or near-continuous growth phase, theproportionally greater amount of 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine can be produced.

Therefore, the invention additionally provides a method for producing4-HB, 4-HBal, 4-HBCoA, BDO or putrescine that includes culturing anon-naturally occurring microbial organism having one or more genedisruptions, as disclosed herein. The disruptions can occur in one ormore genes encoding an enzyme that increases production of 4-HB, 4-HBal,4-HBCoA, BDO or putrescine, including optionally coupling 4-HB, 4-HBal,4-HBCoA, BDO or putrescine production to growth of the microorganismwhen the gene disruption reduces or eliminates an activity of theenzyme.

In some embodiments, the gene disruption can include a complete genedeletion. In some embodiments other methods to disrupt a gene include,for example, frameshifting by omission or addition of oligonucleotidesor by mutations that render the gene inoperable. One skilled in the artwill recognize the advantages of gene deletions, however, because of thestability it confers to the non-naturally occurring organism fromreverting to a parental phenotype in which the gene disruption has notoccurred. In particular, the gene disruptions are selected from the genesets as disclosed herein.

Once computational or other predictions are made of one or more genessets disruption to increase production of 4-HB, 4-HBal, 4-HBCoA, BDO orputrescine, the strains can be constructed, evolved, and tested. Genedisruptions, including gene deletions, are introduced into host organismby methods well known in the art. A particularly useful method for genedisruption is by homologous recombination, as disclosed herein.

The engineered strains can be characterized by measuring the growthrate, the substrate uptake rate, and/or the product/byproduct secretionrate. Cultures can be grown and used as inoculum for a fresh batchculture for which measurements are taken during exponential growth. Thegrowth rate can be determined by measuring optical density using aspectrophotometer (A600). Concentrations of glucose and other organicacid byproducts in the culture supernatant can be determined by wellknown methods such as HPLC, GC-MS or other well known analytical methodssuitable for the analysis of the desired product, as disclosed herein,and used to calculate uptake and secretion rates.

Strains containing gene disruptions can exhibit suboptimal growth ratesuntil their metabolic networks have adjusted to their missingfunctionalities. To assist in this adjustment, the strains can beadaptively evolved. By subjecting the strains to adaptive evolution,cellular growth rate becomes the primary selection pressure and themutant cells are compelled to reallocate their metabolic fluxes in orderto enhance their rates of growth. This reprogramming of metabolism hasbeen recently demonstrated for several E. coli mutants that had beenadaptively evolved on various substrates to reach the growth ratespredicted a priori by an in silico model (Fong and Palsson, Nat. Genet.36:1056-1058 (2004)). The growth improvements brought about by adaptiveevolution can be accompanied by enhanced rates of 4-HB, 4-HBal, 4-HBCoA,BDO or putrescine production. The strains can optionally be adaptivelyevolved in replicate, running in parallel, to account for differences inthe evolutionary patterns that can be exhibited by a host organism (Fongand Palsson, Nat. Genet. 36:1056-1058 (2004); Fong et al., J. Bacteriol.185:6400-6408 (2003); Ibarra et al., Nature 420:186-189 (2002)) thatcould potentially result in one strain having superior productionqualities over the others. Evolutions can be run for a period of time,typically 2-6 weeks, depending upon the rate of growth improvementattained. In general, evolutions are stopped once a stable phenotype isobtained.

Following the adaptive evolution process, the new strains arecharacterized again by measuring the growth rate, the substrate uptakerate, and the product/byproduct secretion rate. These results arecompared to the theoretical predictions by plotting actual growth andproduction yields along side the production envelopes from metabolicmodeling. The most successful design/evolution combinations are chosento pursue further, and are characterized in lab-scale batch andcontinuous fermentations. The growth-coupled biochemical productionconcept behind the methods disclosed herein such as OptKnock approachshould also result in the generation of genetically stableoverproducers. Thus, the cultures are maintained in continuous mode foran extended period of time, for example, one month or more, to evaluatelong-term stability. Periodic samples can be taken to ensure that yieldand productivity are maintained.

Adaptive evolution is a powerful technique that can be used to increasegrowth rates of mutant or engineered microbial strains, or of wild-typestrains growing under unnatural environmental conditions. It isespecially useful for strains designed via methods such as OptKnock,which results in growth-coupled product formation. Therefore, evolutiontoward optimal growing strains will indirectly optimize production aswell. Unique strains of E. coli K-12 MG1655 were created through geneknockouts and adaptive evolution. (Fong and Palsson, Nat. Genet.36:1056-1058 (2004)). In this work, all adaptive evolutionary cultureswere maintained in prolonged exponential growth by serial passage ofbatch cultures into fresh medium before the stationary phase wasreached, thus rendering growth rate as the primary selection pressure.Knockout strains were constructed and evolved on minimal mediumsupplemented with different carbon substrates (four for each knockoutstrain). Evolution cultures were carried out in duplicate or triplicate,giving a total of 50 evolution knockout strains. The evolution cultureswere maintained in exponential growth until a stable growth rate wasreached. The computational predictions were accurate (within 10%) atpredicting the post-evolution growth rate of the knockout strains in 38out of the 50 cases examined. Furthermore, a combination of OptKnockdesign with adaptive evolution has led to improved lactic acidproduction strains. (Fong et al., Biotechnol. Bioeng. 91:643-648(2005)). Similar methods can be applied to the strains disclosed hereinand applied to various host strains.

There are a number of developed technologies for carrying out adaptiveevolution. Exemplary methods are disclosed herein. In some embodiments,optimization of a non-naturally occurring organism of the presentinvention includes utilizing adaptive evolution techniques to increase4-HB, 4-HBal, 4-HBCoA, BDO or putrescine production and/or stability ofthe producing strain.

Serial culture involves repetitive transfer of a small volume of grownculture to a much larger vessel containing fresh growth medium. When thecultured organisms have grown to saturation in the new vessel, theprocess is repeated. This method has been used to achieve the longestdemonstrations of sustained culture in the literature (Lenski andTravisano, Proc. Natl. Acad. Sci. USA 91:6808-6814 (1994)) inexperiments which clearly demonstrated consistent improvement inreproductive rate over a period of years. Typically, transfer ofcultures is usually performed during exponential phase, so each day thetransfer volume is precisely calculated to maintain exponential growththrough the next 24 hour period. Manual serial dilution is inexpensiveand easy to parallelize.

In continuous culture the growth of cells in a chemostat represents anextreme case of dilution in which a very high fraction of the cellpopulation remains. As a culture grows and becomes saturated, a smallproportion of the grown culture is replaced with fresh media, allowingthe culture to continually grow at close to its maximum population size.Chemostats have been used to demonstrate short periods of rapidimprovement in reproductive rate (Dykhuizen, Methods Enzymol. 613-631(1993)). The potential usefulness of these devices was recognized, buttraditional chemostats were unable to sustain long periods of selectionfor increased reproduction rate, due to the unintended selection ofdilution-resistant (static) variants. These variants are able to resistdilution by adhering to the surface of the chemostat, and by doing so,outcomplete less adherent individuals, including those that have higherreproductive rates, thus obviating the intended purpose of the device(Chao and Ramsdell, J. Gen. Microbiol. 20:132-138 (1985)). One possibleway to overcome this drawback is the implementation of a device with twogrowth chambers, which periodically undergo transient phases ofsterilization, as described previously (Marliere and Mutzel, U.S. Pat.No. 6,686,194).

Evolugator™ is a continuous culture device developed by Evolugate, LLC(Gainesville, Fla.) and exhibits significant time and effort savingsover traditional evolution techniques (de Crecy et al., Appl. Microbiol.Biotechnol. 77:489-496 (2007)). The cells are maintained in prolongedexponential growth by the serial passage of batch cultures into freshmedium before the stationary phase is attained. By automating opticaldensity measurement and liquid handling, the Evolugator™ can performserial transfer at high rates using large culture volumes, thusapproaching the efficiency of a chemostat in evolution of cell fitness.For example, a mutant of Acinetobacter sp ADP1 deficient in a componentof the translation apparatus, and having severely hampered growth, wasevolved in 200 generations to 80% of the wild-type growth rate. However,in contrast to the chemostat which maintains cells in a single vessel,the machine operates by moving from one “reactor” to the next insubdivided regions of a spool of tubing, thus eliminating any selectionfor wall-growth. The transfer volume is adjustable, and normally set toabout 50%. A drawback to this device is that it is large and costly,thus running large numbers of evolutions in parallel is not practical.Furthermore, gas addition is not well regulated, and strict anaerobicconditions are not maintained with the current device configuration.Nevertheless, this is an alternative method to adaptively evolve aproduction strain.

As disclosed herein, a nucleic acid encoding a desired activity of a4-HB, 4-HBal, 4-HBCoA, BDO or putrescine pathway can be introduced intoa host organism. In some cases, it can be desirable to modify anactivity of a 4-HB, 4-HBal, 4-HBCoA BDO or putrescine pathway enzyme orprotein to increase production of 4-HB, 4-HBal, 4-HBCoA BDO orputrescine. For example, known mutations that increase the activity of aprotein or enzyme can be introduced into an encoding nucleic acidmolecule. Additionally, optimization methods can be applied to increasethe activity of an enzyme or protein and/or decrease an inhibitoryactivity, for example, decrease the activity of a negative regulator.

One such optimization method is directed evolution. Directed evolutionis a powerful approach that involves the introduction of mutationstargeted to a specific gene in order to improve and/or alter theproperties of an enzyme. Improved and/or altered enzymes can beidentified through the development and implementation of sensitivehigh-throughput screening assays that allow the automated screening ofmany enzyme variants (for example, >10⁴). Iterative rounds ofmutagenesis and screening typically are performed to afford an enzymewith optimized properties. Computational algorithms that can help toidentify areas of the gene for mutagenesis also have been developed andcan significantly reduce the number of enzyme variants that need to begenerated and screened. Numerous directed evolution technologies havebeen developed (for reviews, see Hibbert et al., Biomol. Eng 22:11-19(2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical andbiotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press;Otten and Quax. Biomol. Eng 22:1-9 (2005).; and Sen et al., ApplBiochem. Biotechnol 143:212-223 (2007)) to be effective at creatingdiverse variant libraries, and these methods have been successfullyapplied to the improvement of a wide range of properties across manyenzyme classes. Enzyme characteristics that have been improved and/oraltered by directed evolution technologies include, for example:selectivity/specificity, for conversion of non-natural substrates;temperature stability, for robust high temperature processing; pHstability, for bioprocessing under lower or higher pH conditions;substrate or product tolerance, so that high product titers can beachieved; binding (K_(m)), including broadening substrate binding toinclude non-natural substrates; inhibition (K_(i)), to remove inhibitionby products, substrates, or key intermediates; activity (kcat), toincreases enzymatic reaction rates to achieve desired flux; expressionlevels, to increase protein yields and overall pathway flux; oxygenstability, for operation of air sensitive enzymes under aerobicconditions; and anaerobic activity, for operation of an aerobic enzymein the absence of oxygen.

A number of exemplary methods have been developed for the mutagenesisand diversification of genes to target desired properties of specificenzymes. Such methods are well known to those skilled in the art. Any ofthese can be used to alter and/or optimize the activity of a 4-HB,4-HBal, 4-HBCoA, BDO or putrescine pathway enzyme or protein. Suchmethods include, but are not limited to EpPCR, which introduces randompoint mutations by reducing the fidelity of DNA polymerase in PCRreactions (Pritchard et al., J. Theor. Biol. 234:497-509 (2005));Error-prone Rolling Circle Amplification (epRCA), which is similar toepPCR except a whole circular plasmid is used as the template and random6-mers with exonuclease resistant thiophosphate linkages on the last 2nucleotides are used to amplify the plasmid followed by transformationinto cells in which the plasmid is re-circularized at tandem repeats(Fujii et al., Nucleic Acids Res. 32:e145 (2004); and Fujii et al., Nat.Protoc. 1:2493-2497 (2006)); DNA or Family Shuffling, which typicallyinvolves digestion of two or more variant genes with nucleases such asDnase I or EndoV to generate a pool of random fragments that arereassembled by cycles of annealing and extension in the presence of DNApolymerase to create a library of chimeric genes (Stemmer, Proc NatlAcad Sci USA 91:10747-10751 (1994); and Stemmer, Nature 370:389-391(1994)); Staggered Extension (StEP), which entails template primingfollowed by repeated cycles of 2 step PCR with denaturation and veryshort duration of annealing/extension (as short as 5 sec) (Zhao et al.,Nat. Biotechnol. 16:258-261 (1998)); Random Priming Recombination (RPR),in which random sequence primers are used to generate many short DNAfragments complementary to different segments of the template (Shao etal., Nucleic Acids Res 26:681-683 (1998)).

Additional methods include Heteroduplex Recombination, in whichlinearized plasmid DNA is used to form heteroduplexes that are repairedby mismatch repair (Volkov et al, Nucleic Acids Res. 27:e18 (1999); andVolkov et al., Methods Enzymol. 328:456-463 (2000)); RandomChimeragenesis on Transient Templates (RACHITT), which employs Dnase Ifragmentation and size fractionation of single stranded DNA (ssDNA)(Coco et al., Nat. Biotechnol. 19:354-359 (2001)); Recombined Extensionon Truncated templates (RETT), which entails template switching ofunidirectionally growing strands from primers in the presence ofunidirectional ssDNA fragments used as a pool of templates (Lee et al.,J. Molec. Catalysis 26:119-129 (2003)); Degenerate Oligonucleotide GeneShuffling (DOGS), in which degenerate primers are used to controlrecombination between molecules; (Bergquist and Gibbs, Methods Mol.Biol. 352:191-204 (2007); Bergquist et al., Biomol. Eng 22:63-72 (2005);Gibbs et al., Gene 271:13-20 (2001)); Incremental Truncation for theCreation of Hybrid Enzymes (ITCHY), which creates a combinatoriallibrary with 1 base pair deletions of a gene or gene fragment ofinterest (Ostermeier et al., Proc. Natl. Acad. Sci. USA 96:3562-3567(1999); and Ostermeier et al., Nat. Biotechnol. 17:1205-1209 (1999));Thio-Incremental Truncation for the Creation of Hybrid Enzymes(THIO-ITCHY), which is similar to ITCHY except that phosphothioate dNTPsare used to generate truncations (Lutz et al., Nucleic Acids Res 29:E16(2001)); SCRATCHY, which combines two methods for recombining genes,ITCHY and DNA shuffling (Lutz et al., Proc. Natl. Acad. Sci. USA98:11248-11253 (2001)); Random Drift Mutagenesis (RNDM), in whichmutations made via epPCR are followed by screening/selection for thoseretaining usable activity (Bergquist et al., Biomol. Eng. 22:63-72(2005)); Sequence Saturation Mutagenesis (SeSaM), a random mutagenesismethod that generates a pool of random length fragments using randomincorporation of a phosphothioate nucleotide and cleavage, which is usedas a template to extend in the presence of “universal” bases such asinosine, and replication of an inosine-containing complement givesrandom base incorporation and, consequently, mutagenesis (Wong et al.,Biotechnol. J. 3:74-82 (2008); Wong et al., Nucleic Acids Res. 32:e26(2004); and Wong et al., Anal. Biochem. 341:187-189 (2005)); SyntheticShuffling, which uses overlapping oligonucleotides designed to encode“all genetic diversity in targets” and allows a very high diversity forthe shuffled progeny (Ness et al., Nat. Biotechnol. 20:1251-1255(2002)); Nucleotide Exchange and Excision Technology NexT, whichexploits a combination of dUTP incorporation followed by treatment withuracil DNA glycosylase and then piperidine to perform endpoint DNAfragmentation (Muller et al., Nucleic Acids Res. 33:e117 (2005)).

Further methods include Sequence Homology-Independent ProteinRecombination (SHIPREC), in which a linker is used to facilitate fusionbetween two distantly related or unrelated genes, and a range ofchimeras is generated between the two genes, resulting in libraries ofsingle-crossover hybrids (Sieber et al., Nat. Biotechnol. 19:456-460(2001)); Gene Site Saturation Mutagenesis™ (GSSM™), in which thestarting materials include a supercoiled double stranded DNA (dsDNA)plasmid containing an insert and two primers which are degenerate at thedesired site of mutations (Kretz et al., Methods Enzymol. 388:3-11(2004)); Combinatorial Cassette Mutagenesis (CCM), which involves theuse of short oligonucleotide cassettes to replace limited regions with alarge number of possible amino acid sequence alterations (Reidhaar-Olsonet al. Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al.Science 241:53-57 (1988)); Combinatorial Multiple Cassette Mutagenesis(CMCM), which is essentially similar to CCM and uses epPCR at highmutation rate to identify hot spots and hot regions and then extensionby CMCM to cover a defined region of protein sequence space (Reetz etal., Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001)); the MutatorStrains technique, in which conditional is mutator plasmids, utilizingthe mutD5 gene, which encodes a mutant subunit of DNA polymerase III, toallow increases of 20 to 4000-X in random and natural mutation frequencyduring selection and block accumulation of deleterious mutations whenselection is not required (Selifonova et al., Appl. Environ. Microbiol.67:3645-3649 (2001)); Low et al., J. Mol. Biol. 260:359-3680 (1996)).

Additional exemplary methods include Look-Through Mutagenesis (LTM),which is a multidimensional mutagenesis method that assesses andoptimizes combinatorial mutations of selected amino acids (Rajpal etal., Proc. Natl. Acad. Sci. USA 102:8466-8471 (2005)); Gene Reassembly,which is a DNA shuffling method that can be applied to multiple genes atone time or to create a large library of chimeras (multiple mutations)of a single gene (Tunable GeneReassembly™ (TGR™) Technology supplied byVerenium Corporation), in Silico Protein Design Automation (PDA), whichis an optimization algorithm that anchors the structurally definedprotein backbone possessing a particular fold, and searches sequencespace for amino acid substitutions that can stabilize the fold andoverall protein energetics, and generally works most effectively onproteins with known three-dimensional structures (Hayes et al., Proc.Natl. Acad. Sci. USA 99:15926-15931 (2002)); and Iterative SaturationMutagenesis (ISM), which involves using knowledge of structure/functionto choose a likely site for enzyme improvement, performing saturationmutagenesis at chosen site using a mutagenesis method such as StratageneQuikChange (Stratagene; San Diego Calif.), screening/selecting fordesired properties, and, using improved clone(s), starting over atanother site and continue repeating until a desired activity is achieved(Reetz et al., Nat. Protoc. 2:891-903 (2007); and Reetz et al., Angew.Chem. Int. Ed Engl. 45:7745-7751 (2006)).

Any of the aforementioned methods for mutagenesis can be used alone orin any combination. Additionally, any one or combination of the directedevolution methods can be used in conjunction with adaptive evolutiontechniques, as described herein.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I Biosynthesis of 4-Hydroxybutanoic Acid

This example describes exemplary biochemical pathways for 4-HBproduction.

Previous reports of 4-HB synthesis in microbes have focused on thiscompound as an intermediate in production of the biodegradable plasticpoly-hydroxyalkanoate (PHA) (U.S. Pat. No. 6,117,658). The use of4-HB/3-HB copolymers over poly-3-hydroxybutyrate polymer (PHB) canresult in plastic that is less brittle (Saito and Doi, Intl. J. Biol.Macromol. 16:99-104 (1994)). The production of monomeric 4-HB describedherein is a fundamentally distinct process for several reasons: (1) theproduct is secreted, as opposed to PHA which is produced intracellularlyand remains in the cell; (2) for organisms that produce hydroxybutanoatepolymers, free 4-HB is not produced, but rather the Coenzyme Aderivative is used by the polyhydroxyalkanoate synthase; (3) in the caseof the polymer, formation of the granular product changesthermodynamics; and (4) extracellular pH is not an issue for productionof the polymer, whereas it will affect whether 4-HB is present in thefree acid or conjugate base state, and also the equilibrium between 4-HBand GBL.

4-HB can be produced in two enzymatic reduction steps from succinate, acentral metabolite of the TCA cycle, with succinic semialdehyde as theintermediate (FIG. 1). The first of these enzymes, succinic semialdehydedehydrogenase, is native to many organisms including E. coli, in whichboth NADH- and NADPH-dependent enzymes have been found (Donnelly andCooper, Eur. J. Biochem. 113:555-561 (1981); Donnelly and Cooper, J.Bacteriol. 145:1425-1427 (1981); Marek and Henson, J.Bacteriol.170:991-994 (1988)). There is also evidence supportingsuccinic semialdehyde dehydrogenase activity in S. cerevisiae (Ramos etal., Eur. J. Biochem. 149:401-404 (1985)), and a putative gene has beenidentified by sequence homology. However, most reports indicate thatthis enzyme proceeds in the direction of succinate synthesis, as shownin FIG. 1 (Donnelly and Cooper, supra; Lutke-Eversloh and Steinbuchel,FEMS Microbiol. Lett. 181:63-71 (1999)), participating in thedegradation pathway of 4-HB and gamma-aminobutyrate. Succinicsemialdehyde also is natively produced by certain microbial organismssuch as E. coli through the TCA cycle intermediate α-ketogluterate viathe action of two enzymes: glutamate:succinic semialdehyde transaminaseand glutamate decarboxylase. An alternative pathway, used by theobligate anaerobe Clostridium kluyveri to degrade succinate, activatessuccinate to succinyl-CoA, then converts succinyl-CoA to succinicsemialdehyde using an alternative succinic semialdehyde dehydrogenasewhich is known to function in this direction (Sohling and Gottschalk,Eur. J. Biochem. 212:121-127 (1993)). However, this route has theenergetic cost of ATP required to convert succinate to succinyl-CoA.

The second enzyme of the pathway, 4-hydroxybutanoate dehydrogenase, isnot native to E. coli or yeast but is found in various bacteria such asC. kluyveri and Ralstonia eutropha (Lutke-Eversloh and Steinbuchel,supra; Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996);Valentin et al., Eur. J. Biochem. 227:43-60 (1995); Wolff and Kenealy,Protein Expr. Pur 6:206-212 (1995)). These enzymes are known to beNADH-dependent, though NADPH-dependent forms also exist. An additionalpathway to 4-HB from alpha-ketoglutarate was demonstrated in E. coliresulting in the accumulation of poly(4-hydroxybutyric acid) (Song etal., Wei Sheng Wu Xue. Bao. 45:382-386 (2005)). The recombinant strainrequired the overexpression of three heterologous genes, PHA synthase(R. eutropha), 4-hydroxybutyrate dehydrogenase (R. eutropha) and4-hydroxybutyrate:CoA transferase (C. kluyveri), along with two nativeE. coli genes: glutamate:succinic semialdehyde transaminase andglutamate decarboxylase. Steps 4 and 5 in FIG. 1 can alternatively becarried out by an alpha-ketoglutarate decarboxylase such as the oneidentified in Euglena gracilis (Shigeoka et al., Biochem. J.282(Pt2):319-323 (1992); Shigeoka and Nakano, Arch. Biochem. Biophys.288:22-28 (1991); Shigeoka and Nakano, Biochem J. 292(Pt 2):463-467(1993)). However, this enzyme has not previously been applied to impactthe production of 4-HB or related polymers in any organism.

The microbial production capabilities of 4-hydroxybutyrate were exploredin two microbes, Escherichia coli and Saccharomyces cerevisiae, using insilico metabolic models of each organism. Potential pathways to 4-HBproceed via a succinate, succinyl-CoA, or alpha-ketoglutarateintermediate as shown in FIG. 1.

A first step in the 4-HB production pathway from succinate involves theconversion of succinate to succinic semialdehyde via an NADH- orNADPH-dependant succinic semialdehyde dehydrogenase. In E. coli, gabD isan NADP-dependant succinic semialdehyde dehydrogenase and is part of agene cluster involved in 4-aminobutyrate uptake and degradation(Niegemann et al., Arch. Microbiol. 160:454-460 (1993); Schneider etal., J. Bacteriol. 184:6976-6986 (2002)). sad is believed to encode theenzyme for NAD-dependant succinic semialdehyde dehydrogenase activity(Marek and Henson, supra). S. cerevisiae contains only theNADPH-dependant succinic semialdehyde dehydrogenase, putatively assignedto UGA2, which localizes to the cytosol (Huh et al., Nature 425:686-691(2003)). The maximum yield calculations assuming the succinate pathwayto 4-HB in both E. coli and S. cerevisiae require only the assumptionthat a non-native 4-HB dehydrogenase has been added to their metabolicnetworks.

The pathway from succinyl-CoA to 4-hydroxybutyrate was described in U.S.Pat. No. 6,117,658 as part of a process for making polyhydroxyalkanoatescomprising 4-hydroxybutyrate monomer units. Clostridium kluyveri is oneexample organism known to possess CoA-dependant succinic semialdehydedehydrogenase activity (Sohling and Gottschalk, supra; Sohling andGottschalk, supra). In this study, it is assumed that this enzyme, fromC. kluyveri or another organism, is expressed in E. coli or S.cerevisiae along with a non-native or heterologous 4-HB dehydrogenase tocomplete the pathway from succinyl-CoA to 4-HB. The pathway fromalpha-ketoglutarate to 4-HB was demonstrated in E. coli resulting in theaccumulation of poly(4-hydroxybutyric acid) to 30% of dry cell weight(Song et al., supra). As E. coli and S. cerevisiae natively orendogenously possess both glutamate:succinic semialdehyde transaminaseand glutamate decarboxylase (Coleman et al., J. Biol. Chem. 276:244-250(2001)), the pathway from AKG to 4-HB can be completed in both organismsby assuming only that a non-native 4-HB dehydrogenase is present.

EXAMPLE II Biosynthesis of 1,4-Butanediol from Succinate andAlpha-ketoglutarate

This example illustrates the construction and biosynthetic production of4-HB and BDO from microbial organisms. Pathways for 4-HB and BDO aredisclosed herein.

There are several alternative enzymes that can be utilized in thepathway described above. The native or endogenous enzyme for conversionof succinate to succinyl-CoA (Step 1 in FIG. 1) can be replaced by a CoAtransferase such as that encoded by the cat1 gene C. kluyveri (Sohlingand Gottschalk, Eur. J Biochem. 212:121-127 (1993)), which functions ina similar manner to Step 9. However, the production of acetate by thisenzyme may not be optimal, as it might be secreted rather than beingconverted back to acetyl-CoA. In this respect, it also can be beneficialto eliminate acetate formation in Step 9. As one alternative to this CoAtransferase, a mechanism can be employed in which the 4-HB is firstphosphorylated by ATP and then converted to the CoA derivative, similarto the acetate kinase/phosphotransacetylase pathway in E. coli for theconversion of acetate to acetyl-CoA. The net cost of this route is oneATP, which is the same as is required to regenerate acetyl-CoA fromacetate. The enzymes phosphotransbutyrylase (ptb) and butyrate kinase(bk) are known to carry out these steps on the non-hydroxylatedmolecules for butyrate production in C. acetobutylicum (Cary et al.,Appl Environ Microbiol 56:1576-1583 (1990); Valentine, R. C. and R. S.Wolfe, J Biol. Chem. 235:1948-1952 (1960)). These enzymes arereversible, allowing synthesis to proceed in the direction of 4-HB.

BDO also can be produced via α-ketoglutarate in addition to or insteadof through succinate. A described previously, and exemplified furtherbelow, one pathway to accomplish product biosynthesis is with theproduction of succinic semialdehyde via α-ketoglutarate using theendogenous enzymes (FIG. 1, Steps 4-5). An alternative is to use anα-ketoglutarate decarboxylase that can perform this conversion in onestep (FIG. 1, Step 8; Tian et al., Proc Natl Acad Sci U S.A102:10670-10675 (2005)).

For the construction of different strains of BDO-producing microbialorganisms, a list of applicable genes was assembled for corroboration.Briefly, one or more genes within the 4-HB and/or BDO biosyntheticpathways were identified for each step of the complete BDO-producingpathway shown in FIG. 1, using available literature resources, the NCBIgenetic database, and homology searches. The genes cloned and assessedin this study are presented below in in Table 6, along with theappropriate references and URL citations to the polypeptide sequence. Asdiscussed further below, some genes were synthesized for codonoptimization while others were cloned via PCR from the genomic DNA ofthe native or wild-type organism. For some genes both approaches wereused, and in this case the native genes are indicated by an “n” suffixto the gene identification number when used in an experiment. Note thatonly the DNA sequences differ; the proteins are identical.

TABLE 6 Genes expressed in host BDO-producting microbial organisms.Reaction Gene ID number Gene number (FIG. 1) name Source organism Enzymename Link to protein sequence Reference 0001 9 Cat2 Clostridium kluyveri4-hydroxybutyrate www.ncbi.nlm.nih.gov/entrez/ 1 DSM 555 coenzyme Aviewer.fcgi?db=nuccore&id= transferase 1228100 0002 12/13 adhEClostridium acetobutylicum Aldehyde/alcohol www.ncbi.nlm.nih.gov/entrez/2 ATCC 824 dehydrogenase viewer.fcgi?db=protein&val= 15004739 0003 12/13adhE2 Clostridium acetobutylicum Aldehyde/alcoholwww.ncbi.nlm.nih.gov/entrez/ 2 ATCC 824 dehydrogenaseviewer.fcgi?val=NP_149325.1 0004 1 Cat1 Clostridium kluyveri Succinatewww.ncbi.nlm.nih.gov/entrez/ 1 DSM 555 coenzyme Aviewer.fcgi?db=nuccore&id= transferase 1228100 0008 6 sucD Clostridiumkluyveri Succinic www.ncbi.nlm.nih.gov/entrez/ 1 DSM 555 semialdehydeviewer.fcgi?db=nuccore&id= dehydrogenase 1228100 (CoA-dependent) 0009 74-HBd Ralstonia eutropha 4-hydroxybutyrate www.ncbi.nlm.nih.gov/entrez/2 H16 dehydrogenase viewer.fcgi?val=YP_726053.1 (NAD-dependent) 0010 74-HBd Clostridium kluyveri 4-hydroxybutyratewww.ncbi.nlm.nih.gov/entrez/ 1 DSM 555 dehydrogenaseviewer.fcgi?db=nuccore&id= (NAD-dependent) 1228100 0011 12/13 adhE E.coli Aldehyde/alcohol www.shigen.nig.ac.jp/ecoli/pec/ dehydrogenasegenes.List.DetailAction.do? fromListFlag=true&featureType= 1&orfId=12190012 12/13 yqhD E. coli Aldehyde/alcohol www.shigen.nig.ac.jp/ecoli/pec/dehydrogenase genes.List.DetailAction.do 0013 13 bdhB Clostridiumacetobutylicum Butanol www.ncbi.nlm.nih.gov/entrez/ 2 ATCC 824dehydrogenase II viewer.fcgi?val=NP_349891.1 0020 11 ptb Clostridiumacetobutylicum Phospho- www.ncbi.nlm.nih.gov/entrez/ 2 ATCC 824transbutyrylase viewer.fcgi?db=protein&id= 15896327 0021 10 buk1Clostridium acetobutylicum Butyrate kinase Iwww.ncbi.nlm.nih.gov/entrez/ 2 ATCC 824 viewer.fcgi?db=protein&id=20137334 0022 10 buk2 Clostridium acetobutylicum Butyrate kinase IIwww.ncbi.nlm.nih.gov/entrez/ 2 ATCC 824 viewer.fcgi?db=protein&id=20137415 0023 13 adhEm isolated from metalibrary Alcohol (37)d} ofanaerobic sewage digester dehydrogenase microbial consortia 0024 13 adhEClostridium thermocellum Alcohol www.genome.jp/dbget- dehydrogenasebin/www_bget?cth:Cthe_0423 0025 13 ald Clostridium beijerinckii CoenzymeA- www.ncbi.nlm.nih.gov/entrez/ (31)d} acylating aldehydeviewer.fcgi?db=protein&id= dehydrogenase 49036681 0026 13 bdhAClostridium acetobutylicum Butanol www.ncbi.nlm.nih.gov/entrez/ 2 ATCC824 dehydrogenase viewer.fcgi?val=NP_349892.1 0027 12 bld ClostridiumButyraldehyde www.ncbi.nlm.nih.gov/entrez/ 4 saccharoperbutylacetonicumdehydrogenase viewer.fcgi?db=protein&id= 31075383 0028 13 bdhClostridium Butanol www.ncbi.nlm.nih.gov/entrez/ 4saccharoperbutylacetonicum dehydrogenase viewer.fcgi?db=protein&id=124221917 0029 12/13 adhE Clostridium tetani Aldehyde/alcoholwww.genome.jp/dbget- dehydrogenase bin/www_bget?ctc:CTC01366 0030 12/13adhE Clostridium perfringens Aldehyde/alcohol www.genome.jp/dbget-dehydrogenase bin/www_bget?cpe:CPE2531 0031 12/13 adhE Clostridiumdifficile Aldehyde/alcohol www.genome.jp/dbget- dehydrogenasebin/www_bget?cdf:CD2966 0032 8 sucA Mycobacterium bovis α-ketoglutaratewww.ncbi.nlm.nih.gov/entrez/ 5 BCG, Pasteur decarboxylaseviewer.fcgi?val=YP_977400.1 0033 9 cat2 Clostridium aminobutyricum4-hydroxybutyrate www.ncbi.nlm.nih.gov/entrez/ coenzyme Aviewer.fcgi?db=protein&val= transferase 6249316 0034 9 cat2Porphyromonas gingivalis 4-hydroxybutyrate www.ncbi.nlm.nih.gov/entrez/W83 coenzyme A viewer.fcgi?db=protein&val= transferase 34541558 0035 6sucD Porphyromonas gingivalis Succinic www.ncbi.nlm.nih.gov/entrez/ W83semialdehyde viewer.fcgi?val=NP_904963.1 dehydrogenase (CoA-dependent)0036 7 4-HBd Porphyromonas gingivalis NAD-dependentwww.ncbi.nlm.nih.gov/entrez/ W83 4-hydroxybutyrateviewer.fcgi?val=NP_904964.1 dehydrogenase 0037 7 gbd Unculturedbacterium 4-hydroxybutyrate www.ncbi.nlm.nih.gov/entrez/ 6 dehydrogenaseviewer.fcgi?db=nuccore&id= 5916168 0038 1 sucCD E. coli Succinyl-CoAwww.shigen.nig.ac.jp/ecoli/pec/ synthetase genes.List.DetailAction.do 1Sohling and Gottschalk, Eur. J. Biochem. 212: 121-127 (1993); Sohlingand Gottschalk, J. Bacteriol. 178: 871-880 (1996) 2 Nolling et al., J.,J. Bacteriol. 183: 4823-4838 (2001) 3 Pohlmann et al., Nat. Biotechnol.24: 1257-1262 (2006) 4 Kosaka et al., Biosci. Biotechnol. Biochem. 71:58-68 (2007) 5 Brosch et al., Proc. Natl. Acad. Sci. U.S.A. 104:5596-5601 (2007) 6 Henne et al., Appl. Environ. Microbiol. 65: 3901-3907(1999)

Expression Vector Construction for BDO pathway. Vector backbones andsome strains were obtained from Dr. Rolf Lutz of Expressys(expressys.de/). The vectors and strains are based on the pZ ExpressionSystem developed by Dr. Rolf Lutz and Prof. Hermann Bujard (Lutz, R. andH. Bujard, Nucleic Acids Res 25:1203-1210 (1997)). Vectors obtained werepZE13luc, pZA33luc, pZS*13luc and pZE22luc and contained the luciferasegene as a stuffer fragment. To replace the luciferase stuffer fragmentwith a lacZ-alpha fragment flanked by appropriate restriction enzymesites, the luciferase stuffer fragment was first removed from eachvector by digestion with EcoRI and XbaI. The lacZ-alpha fragment was PCRamplified from pUC19 with the following primers:

lacZalpha-RI (SEQ ID NO: 1)5′GACGAATTCGCTAGCAAGAGGAGAAGTCGACATGTCCAATTCACTGGC CGTCGTTTTAC3′lacZalpha 3′BB (SEQ ID NO: 2)5′-GACCCTAGGAAGCTTTCTAGAGTCGACCTATGCGGCATCAGAGCAG A-3′.

This generated a fragment with a 5′ end of EcoRI site, NheI site, aRibosomal Binding Site, a SalI site and the start codon. On the 3′ endof the fragment contained the stop codon, XbaI, HindIII, and AvrIIsites. The PCR product was digested with EcoRI and AvrII and ligatedinto the base vectors digested with EcoRI and XbaI (XbaI and AvrII havecompatible ends and generate a non-site). Because NheI and XbaIrestriction enzyme sites generate compatible ends that can be ligatedtogether (but generate a NheI/XbaI non-site that is not digested byeither enzyme), the genes cloned into the vectors could be “Biobricked”together (openwetware.org/wiki/Synthetic_Biology:BioBricks). Briefly,this method allows joining an unlimited number of genes into the vectorusing the same 2 restriction sites (as long as the sites do not appearinternal to the genes), because the sites between the genes aredestroyed after each addition.

All vectors have the pZ designation followed by letters and numbersindication the origin of replication, antibiotic resistance marker andpromoter/regulatory unit. The origin of replication is the second letterand is denoted by E for ColE1, A for p15A and S for pSC101-basedorigins. The first number represents the antibiotic resistance marker (1for Ampicillin, 2 for Kanamycin, 3 for Chloramphenicol, 4 forSpectinomycin and 5 for Tetracycline). The final number defines thepromoter that regulated the gene of interest (1 for P_(LtetO-1), 2 forP_(LlacO-1), 3 for P_(A1lacO-1), and 4 for P_(lac/ara-1)). The MCS andthe gene of interest follows immediately after. For the work discussedhere we employed two base vectors, pZA33 and pZE13, modified for thebiobricks insertions as discussed above. Once the gene(s) of interesthave been cloned into them, resulting plasmids are indicated using thefour digit gene codes given in Table 6; e.g., pZA33-XXXX-YYYY- . . .

Host Strain Construction. The parent strain in all studies describedhere is E. coli K-12 strain MG1655. Markerless deletion strains in adhE,gabD, and aldA were constructed under service contract by a third partyusing the redET method (Datsenko, K. A. and B. L. Wanner, Proc Natl AcadSci U S.A 97:6640-6645 (2000)). Subsequent strains were constructed viabacteriophage P1 mediated transduction (Miller, J. Experiments inMolecular Genetics, Cold Spring Harbor Laboratories, New York (1973)).Strain C600Z1 (laci^(q), PN25-tetR, Sp^(R), lacY1, leuB6,mcrB+, supE44,thi-1, thr-1, tonA21) was obtained from Expressys and was used as asource of a lacIq allele for P1 transduction. Bacteriophage P1vir wasgrown on the C600Z1 E. coli strain, which has the spectinomycinresistance gene linked to the lacI^(q). The P1 lysate grown on C600Z1was used to infect MG1655 with selection for spectinomycin resistance.The spectinomycin resistant colonies were then screened for the linkedlacI^(q) by determining the ability of the transductants to repressexpression of a gene linked to a P_(A1lacO-1) promoter. The resultingstrain was designated MG1655 lacI^(q). A similar procedure was used tointroduce lacI^(Q) into the deletion strains.

Production of 4-HB From Succinate. For construction of a 4-HB producerfrom succinate, genes encoding steps from succinate to 4-HB and 4-HB-CoA(1, 6, 7, and 9 in FIG. 1) were assembled onto the pZA33 and pZE13vectors as described below. Various combinations of genes were assessed,as well as constructs bearing incomplete pathways as controls (Tables 7and 8). The plasmids were then transformed into host strains containinglacI^(Q), which allow inducible expression by addition of isopropylβ-D-1-thiogalactopyranoside (IPTG). Both wild-type and hosts withdeletions in genes encoding the native succinic semialdehydedehydrogenase (step 2 in FIG. 1) were tested.

Activity of the heterologous enzymes were first tested in in vitroassays, using strain MG1655 lacI^(Q) as the host for the plasmidconstructs containing the pathway genes. Cells were grown aerobically inLB media (Difco) containing the appropriate antibiotics for eachconstruct, and induced by addition of IPTG at 1 mM when the opticaldensity (OD600) reached approximately 0.5. Cells were harvested after 6hours, and enzyme assays conducted as discussed below.

In Vitro Enzyme Assays. To obtain crude extracts for activity assays,cells were harvested by centrifugation at 4,500 rpm (Beckman-Coulter,Allegera X-15R) for 10 min. The pellets were resuspended in 0.3 mLBugBuster (Novagen) reagent with benzonase and lysozyme, and lysisproceeded for 15 minutes at room temperature with gentle shaking.Cell-free lysate was obtained by centrifugation at 14,000 rpm (Eppendorfcentrifuge 5402) for 30 min at 4° C. Cell protein in the sample wasdetermined using the method of Bradford et al., Anal. Biochem.72:248-254 (1976), and specific enzyme assays conducted as describedbelow. Activities are reported in Units/mg protein, where a unit ofactivity is defined as the amount of enzyme required to convert 1 mmolof substrate in 1 min. at room temperature. In general, reported valuesare averages of at least 3 replicate assays.

Succinyl-CoA transferase (Cat1) activity was determined by monitoringthe formation of acetyl-CoA from succinyl-CoA and acetate, following apreviously described procedure Sohling and Gottschalk, J. Bacteriol.178:871-880 (1996). Succinyl-CoA synthetase (SucCD) activity wasdetermined by following the formation of succinyl-CoA from succinate andCoA in the presence of ATP. The experiment followed a proceduredescribed by Cha and Parks, J. Biol. Chem. 239:1961-1967 (1964).CoA-dependent succinate semialdehyde dehydrogenase (SucD) activity wasdetermined by following the conversion of NAD to NADH at 340 nm in thepresence of succinate semialdehyde and CoA (Sohling and Gottschalk, Eur.J. Biochem. 212:121-127 (1993)). 4-HB dehydrogenase (4-HBd) enzymeactivity was determined by monitoring the oxidation of NADH to NAD at340 nm in the presence of succinate semialdehyde. The experimentfollowed a published procedure Gerhardt et al. Arch. Microbiol.174:189-199 (2000). 4-HB CoA transferase (Cat2) activity was determinedusing a modified procedure from Scherf and Buckel, Appl. Environ.Microbiol. 57:2699-2702 (1991). The formation of 4-HB-CoA or butyryl-CoAformation from acetyl-CoA and 4-HB or butyrate was determined usingHPLC.

Alcohol (ADH) and aldehyde (ALD) dehydrogenase was assayed in thereductive direction using a procedure adapted from several literaturesources (Durre et al., FEMS Microbiol. Rev. 17:251-262 (1995); Palosaariand Rogers, J. Bacteriol. 170:2971-2976 (1988) and Welch et al., Arch.Biochem. Biophys. 273:309-318 (1989). The oxidation of NADH is followedby reading absorbance at 340 nM every four seconds for a total of 240seconds at room temperature. The reductive assays were performed in 100mM MOPS (adjusted to pH 7.5 with KOH), 0.4 mM NADH, and from 1 to 50 μlof cell extract. The reaction is started by adding the followingreagents: 100 μl of 100 mM acetaldehyde or butyraldehyde for ADH, or 100μl of 1 mM acetyl-CoA or butyryl-CoA for ALD. The Spectrophotometer isquickly blanked and then the kinetic read is started. The resultingslope of the reduction in absorbance at 340 nM per minute, along withthe molar extinction coefficient of NAD(P)H at 340 nM (6000) and theprotein concentration of the extract, can be used to determine thespecific activity.

The enzyme activity of PTB is measured in the direction of butyryl-CoAto butyryl-phosphate as described in Cary et al. J. Bacteriol.170:4613-4618 (1988). It provides inorganic phosphate for theconversion, and follows the increase in free CoA with the reagent5,5′-dithiobis-(2-nitrobenzoic acid), or DTNB. DTNB rapidly reacts withthiol groups such as free CoA to release the yellow-colored2-nitro-5-mercaptobenzoic acid (TNB), which absorbs at 412 nm with amolar extinction coefficient of 14,140 M cm⁻¹. The assay buffercontained 150 mM potassium phosphate at pH 7.4, 0.1 mM DTNB, and 0.2 mMbutyryl-CoA, and the reaction was started by addition of 2 to 50 μL cellextract. The enzyme activity of BK is measured in the direction ofbutyrate to butyryl-phosphate formation at the expense of ATP. Theprocedure is similar to the assay for acetate kinase previouslydescribed Rose et al., J. Biol. Chem. 211:737-756 (1954). However it hasbeen found that another acetate kinase enzyme assay protocol provided bySigma to be more useful and sensitive. This assay links conversion ofATP to ADP by acetate kinase to the linked conversion of ADP andphosphoenolpyruvate (PEP) to ATP and pyruvate by pyruvate kinase,followed by the conversion of pyruvate and NADH to lactate and NAD+ bylactate dehydrogenase. Substituting butyrate for acetate is the onlymajor modification to allow the assay to follow BK enzyme activity. Theassay mixture contained 80 mM triethanolamine buffer at pH 7.6, 200 mMsodium butyrate, 10 mM MgCl2, 0.1 mM NADH, 6.6 mM ATP, 1.8 mMphosphoenolpyruvate. Pyruvate kinase, lactate dehydrogenase, andmyokinase were added according to the manufacturer's instructions. Thereaction was started by adding 2 to 50 μL cell extract, and the reactionwas monitored based on the decrease in absorbance at 340 nm indicatingNADH oxidation.

Analysis of CoA Derivatives by HPLC. An HPLC based assay was developedto monitor enzymatic reactions involving coenzyme A (CoA) transfer. Thedeveloped method allowed enzyme activity characterization byquantitative determination of CoA, acetyl CoA (AcCoA), butyryl CoA(BuCoA) and 4-hydroxybutyrate CoA (4-HBCoA) present in in-vitro reactionmixtures. Sensitivity down to low μM was achieved, as well as excellentresolution of all the CoA derivatives of interest.

Chemical and sample preparation was performed as follows. Briefly, CoA,AcCoA, BuCoA and all other chemicals, were obtained from Sigma-Aldrich.The solvents, methanol and acetonitrile, were of HPLC grade. Standardcalibration curves exhibited excellent linearity in the 0.01-1 mg/mLconcentration range. Enzymatic reaction mixtures contained 100 mM TrisHCl buffer (pH 7), aliquots were taken at different time points,quenched with formic acid (0.04% final concentration) and directlyanalyzed by HPLC.

HPLC analysis was performed using an Agilent 1100 HPLC system equippedwith a binary pump, degasser, thermostated autosampler and columncompartment, and diode array detector (DAD), was used for the analysis.A reversed phase column, Kromasil 100 Sum C18, 4.6×150 mm (PeekeScientific), was employed. 25 mM potassium phosphate (pH 7) and methanolor acetonitrile, were used as aqueous and organic solvents at 1 mL/minflow rate. Two methods were developed: a short one with a fastergradient for the analysis of well-resolved CoA, AcCoA and BuCoA, and alonger method for distinguishing between closely eluting AcCoA and4-HBCoA. Short method employed acetonitrile gradient (0 min—5%, 6min—30%, 6.5 min—5%, 10 min—5%) and resulted in the retention times 2.7,4.1 and 5.5 min for CoA, AcCoA and BuCoA, respectively. In the longmethod methanol was used with the following linear gradient: 0 min—5%,20 min—35%, 20.5 min—5%, 25 min—5%. The retention times for CoA, AcCoA,4-HBCoA and BuCoA were 5.8, 8.4, 9.2 and 16.0 min, respectively. Theinjection volume was 5 μL, column temperature 30° C., and UV absorbancewas monitored at 260 nm.

The results demonstrated activity of each of the four pathway steps(Table 7), though activity is clearly dependent on the gene source,position of the gene in the vector, and the context of other genes withwhich it is expressed. For example, gene 0035 encodes a succinicsemialdehyde dehydrogenase that is more active than that encoded by0008, and 0036 and 0010n are more active 4-HB dehydrogenase genes than0009. There also seems to be better 4-HB dehydrogenase activity whenthere is another gene preceding it on the same operon.

TABLE 7 In vitro enzyme activities in cell extracts from MG1655 lacI^(Q)containing the plasmids expressing genes in the 4-HB-CoA pathway.Activities are reported in Units/mg protein, where a unit of activity isdefined as the amount of enzyme required to convert 1 μmol of substratein 1 min. at room temperature. Sample # pZE13 (a) pZA33 (b) OD600 CellProt (c) Cat1 SucD 4HBd Cat2 1 cat1 (0004) 2.71 6.43 1.232 0.00 2 cat1(0004)-sucD (0035) 2.03 5.00 0.761 2.57 3 cat1 (0004)-sucD (0008) 1.043.01 0.783 0.01 4 sucD (0035) 2.31 6.94 2.32 5 sucD (0008) 1.10 4.160.05 6 4hbd (0009) 2.81 7.94 0.003 0.25 7 4hbd (0036) 2.63 7.84 3.31 84hbd (0010n) 2.00 5.08 2.57 9 cat1 (0004)-sucD (0035) 4hbd (0009) 2.075.04 0.600 1.85 0.01 10 cat1 (0004)-sucD (0035) 4hbd (0036) 2.08 5.400.694 1.73 0.41 11 cat1 (0004)-sucD (0035) 4hbd (0010n) 2.44 4.73 0.6792.28 0.37 12 cat1 (0004)-sucD (0008) 4hbd (0009) 1.08 3.99 0.572 −0.010.02 13 cat1 (0004)-sucD (0008) 4hbd (0036) 0.77 2.60 0.898 −0.01 0.0414 cat1 (0004)-sucD (0008) 4hbd (0010n) 0.63 2.47 0.776 0.00 0.00 15cat2 (0034) 2.56 7.86 1.283 16 cat2(0034)-4hbd(0036) 3.13 8.04 24.860.993 17 cat2(0034)-4hbd(0010n) 2.38 7.03 7.45 0.675 184hbd(0036)-cat2(0034) 2.69 8.26 2.15 7.490 19 4hbd(0010n)-cat2(0034)2.44 6.59 0.59 4.101 Genes expressed from Plac on pZE13, a high-copyplasmid with colE1 origin and ampicillin resistance. Gene identificationnumbers are as given in Table 6 Genes expressed from Plac on pZA33, amedium-copy plasmid with pACYC origin and chloramphenicol resistance.(c) Cell protein given as mg protein per mL extract.

Recombinant strains containing genes in the 4-HB pathway were thenevaluated for the ability to produce 4-HB in vivo from central metabolicintermediates. Cells were grown anaerobically in LB medium to OD600 ofapproximately 0.4, then induced with 1 mM IPTG. One hour later, sodiumsuccinate was added to 10 mM, and samples taken for analysis followingan additional 24 and 48 hours. 4-HB in the culture broth was analyzed byGC-MS as described below. The results indicate that the recombinantstrain can produce over 2 mM 4-HB after 24 hours, compared toessentially zero in the control strain (Table 8).

TABLE 8 Production of 4-HB from succinate in E. coli strains harboringplasmids expressing various combinations of 4-HB pathway genes. Sample24 Hours 48 Hours # Host Strain pZE13 pZA33 OD600 4HB, μM 4HB norm. (a)OD600 4HB, μM 4HB norm. (a) 1 MG1655 lacIq cat1 (0004)-sucD (0035) 4hbd(0009) 0.47 487 1036 1.04 1780 1711 2 MG1655 lacIq cat1 (0004)-sucD(0035) 4hbd (0027) 0.41 111 270 0.99 214 217 3 MG1655 lacIq cat1(0004)-sucD (0035) 4hbd (0036) 0.47 863 1835 0.48 2152 4484 4 MG1655lacIq cat1 (0004)-sucD (0035) 4hbd (0010n) 0.46 956 2078 0.49 2221 45335 MG1655 lacIq cat1 (0004)-sucD (0008) 4hbd (0009) 0.38 493 1296 0.371338 3616 6 MG1655 lacIq cat1 (0004)-sucD (0008) 4hbd (0027) 0.32 26 810.27 87 323 7 MG1655 lacIq cat1 (0004)-sucD (0008) 4hbd (0036) 0.24 5062108 0.31 1448 4672 8 MG1655 lacIq cat1 (0004)-sucD (0008) 4hbd (0010n)0.24 78 324 0.56 233 416 9 MG1655 lacIq gabD cat1 (0004)-sucD (0035)4hbd (0009) 0.53 656 1237 1.03 1643 1595 10 MG1655 lacIq gabD cat1(0004)-sucD (0035) 4hbd (0027) 0.44 92 209 0.98 214 218 11 MG1655 lacIqgabD cat1 (0004)-sucD (0035) 4hbd (0036) 0.51 1072 2102 0.97 2358 243112 MG1655 lacIq gabD cat1 (0004)-sucD (0035) 4hbd (0010n) 0.51 981 19240.97 2121 2186 13 MG1655 lacIq gabD cat1 (0004)-sucD (0008) 4hbd (0009)0.35 407 1162 0.77 1178 1530 14 MG1655 lacIq gabD cat1 (0004)-sucD(0008) 4hbd (0027) 0.51 19 36 1.07 50 47 15 MG1655 lacIq gabD cat1(0004)-sucD (0008) 4hbd (0036) 0.35 584 1669 0.78 1350 1731 16 MG1655lacIq gabD cat1 (0004)-sucD (0008) 4hbd (0010n) 0.32 74 232 0.82 232 28317 MG1655 lacIq vector only vector only 0.8 1 2 1.44 3 2 18 MG1655 lacIqgabD vector only vector only 0.89 1 2 1.41 7 5 (a) Normalized 4-HBconcentration, μM/OD600 units

An alternate to using a CoA transferase (cat1) to produce succinyl-CoAfrom succinate is to use the native E. coli sucCD genes, encodingsuccinyl-CoA synthetase. This gene cluster was cloned onto pZE13 alongwith candidate genes for the remaining steps to 4-HB to createpZE13-0038-0035-0036.

Production of 4-HB from Glucose. Although the above experimentsdemonstrate a functional pathway to 4-HB from a central metabolicintermediate (succinate), an industrial process would require theproduction of chemicals from low-cost carbohydrate feedstocks such asglucose or sucrose. Thus, the next set of experiments was aimed todetermine whether endogenous succinate produced by the cells duringgrowth on glucose could fuel the 4-HB pathway. Cells were grownanaerobically in M9 minimal medium (6.78 g/L Na₂HPO₄, 3.0 g/L KH₂PO₄,0.5 g/L NaCl, 1.0 g/L NH₄Cl, 1 mM MgSO₄, 0.1 mM CaCl₂) supplemented with20 g/L glucose, 100 mM 3-(N-morpholino)propanesulfonic acid (MOPS) toimprove the buffering capacity, 10 μg/mL thiamine, and the appropriateantibiotics. 0.25 mM IPTG was added when OD600 reached approximately0.2, and samples taken for 4-HB analysis every 24 hours followinginduction. In all cases 4-HB plateaued after 24 hours, with a maximum ofabout 1 mM in the best strains (FIG. 3a ), while the succinateconcentration continued to rise (FIG. 3b ). This indicates that thesupply of succinate to the pathway is likely not limiting, and that thebottleneck may be in the activity of the enzymes themselves or in NADHavailability. 0035 and 0036 are clearly the best gene candidates forCoA-dependent succinic semialdehyde dehydrogenase and 4-HBdehydrogenase, respectively. The elimination of one or both of the genesencoding known (gabD) or putative (aldA) native succinic semialdehydedehydrogenases had little effect on performance. Finally, it should benoted that the cells grew to a much lower OD in the 4-HB-producingstrains than in the controls (FIG. 3c ).

An alternate pathway for the production of 4-HB from glucose is viaa-ketoglutarate. We explored the use of an α-ketoglutarate decarboxylasefrom Mycobacterium tuberculosis Tian et al., Proc. Natl. Acad. Sci. USA102:10670-10675 (2005) to produce succinic semialdehyde directly fromα-ketoglutarate (step 8 in FIG. 1). To demonstrate that this gene (0032)was functional in vivo, we expressed it on pZE13 in the same host as4-HB dehydrogenase (gene 0036) on pZA33. This strain was capable ofproducing over 1.0 mM 4-HB within 24 hours following induction with 1 mMIPTG (FIG. 4). Since this strain does not express a CoA-dependentsuccinic semialdehyde dehydrogenase, the possibility of succinicsemialdehyde production via succinyl-CoA is eliminated. It is alsopossible that the native genes responsible for producing succinicsemialdehyde could function in this pathway (steps 4 and 5 in FIG. 1);however, the amount of 4-HB produced when the pZE13-0032 plasmid wasleft out of the host is the negligible.

Production of BDO from 4-HB. The production of BDO from 4-HB requiredtwo reduction steps, catalyzed by dehydrogenases. Alcohol and aldehydedehydrogenases (ADH and ALD, respectively) are NAD+/H and/orNADP+/H-dependent enzymes that together can reduce a carboxylic acidgroup on a molecule to an alcohol group, or in reverse, can perform theoxidation of an alcohol to a carboxylic acid. This biotransformation hasbeen demonstrated in wild-type Clostridium acetobutylicum (Jewell etal., Current Microbiology, 13:215-19 (1986)), but neither the enzymesresponsible nor the genes responsible were identified. In addition, itis not known whether activation to 4-HB-CoA is first required (step 9 inFIG. 1), or if the aldehyde dehydrogenase (step 12) can act directly on4-HB. We developed a list of candidate enzymes from C. acetobutylicumand related organisms based on known activity with the non-hydroxylatedanalogues to 4-HB and pathway intermediates, or by similarity to thesecharacterized genes (Table 6). Since some of the candidates aremultifunctional dehydrogenases, they could potentially catalyze both theNAD(P)H-dependent reduction of the acid (or CoA-derivative) to thealdehyde, and of the aldehyde to the alcohol. Before beginning work withthese genes in E. coli, we first validated the result referenced aboveusing C. acetobutylicum ATCC 824. Cells were grown in Schaedler broth(Accumedia, Lansing, Mich.) supplemented with 10 mM 4-HB, in ananaerobic atmosphere of 10% CO₂, 10% H₂, and 80% N₂ at 30° C. Periodicculture samples were taken, centrifuged, and the broth analyzed for BDOby GC-MS as described below. BDO concentrations of 0.1 mM, 0.9 mM, and1.5 mM were detected after 1 day, 2 days, and 7 days incubation,respectively. No BDO was detected in culture grown without 4-HBaddition. To demonstrate that the BDO produced was derived from glucose,we grew the best BDO producing strain MG1655 lacI^(Q)pZE13-0004-0035-0002 pZA33-0034-0036 in M9 minimal medium supplementedwith 4 g/L uniformly labeled ¹³C-glucose. Cells were induced at OD of0.67 with 1 mM IPTG, and a sample taken after 24 hours. Analysis of theculture supernatant was performed by mass spectrometry.

Gene candidates for the 4-HB to BDO conversion pathway were next testedfor activity when expressed in the E. coli host MG1655 lacI^(Q).Recombinant strains containing each gene candidate expressed on pZA33were grown in the presence of 0.25 mM IPTG for four hours at 37° C. tofully induce expression of the enzyme. Four hours after induction, cellswere harvested and assayed for ADH and ALD activity as described above.Since 4-HB-CoA and 4-hydroxybutyraldehyde are not availablecommercially, assays were performed using the non-hydroxylatedsubstrates (Table 9). The ratio in activity between 4-carbon and2-carbon substrates for C. acetobutylicum adhE2 (0002) and E. coli adhE(0011) were similar to those previously reported in the literature aAtsumi et al., Biochim. Biophys. Acta. 1207:1-11 (1994).

TABLE 9 In vitro enzyme activities in cell extracts from MG1655 lacI^(Q)containing pZA33 expressing gene candidates for aldehyde and alcoholdehydrogenases. Aldehyde dehydrogenase Alcohol dehydrogenase SubstrateGene Butyryl-CoA Acetyl-CoA Butyraldehyde Acetaldehyde 0002 0.00760.0046 0.0264 0.0247 0003n 0.0060 0.0072 0.0080 0.0075 0011 0.00690.0095 0.0265 0.0093 0013 N.D. N.D. 0.0130 0.0142 0023 0.0089 0.01370.0178 0.0235 0025 0 0.0001 N.D. N.D. 0026 0 0.0005 0.0024 0.0008Activities are expressed in μmol min⁻¹ mg cell protein⁻¹. N.D., notdetermined.

For the BDO production experiments, cat2 from Porphyromonas gingivalisW83 (gene 0034) was included on pZA33 for the conversion of 4-HB to4-HB-CoA, while the candidate dehydrogenase genes were expressed onpZE13. The host strain was MG1655 lacI^(Q). Along with the alcohol andaldehyde dehydrogenase candidates, we also tested the ability ofCoA-dependent succinic semialdehyde dehydrogenases (sucD) to function inthis step, due to the similarity of the substrates. Cells were grown toan OD of about 0.5 in LB medium supplemented with 10 mM 4-HB, inducedwith 1 mM IPTG, and culture broth samples taken after 24 hours andanalyzed for BDO as described below. The best BDO production occurredusing adhE2 from C. acetobutylicum, sucD from C. kluyveri, or sucD fromP. gingivalis (FIG. 5). Interestingly, the absolute amount of BDOproduced was higher under aerobic conditions; however, this is primarilydue to the lower cell density achieved in anaerobic cultures. Whennormalized to cell OD, the BDO production per unit biomass is higher inanaerobic conditions (Table 10).

TABLE 10 Absolute and normalized BDO concentrations from cultures ofcells expressing adhE2 from C. acetobutylicum, sucD from C. kluyveri, orsucD from P. gingivalis (data from experiments 2, 9, and 10 in FIG. 3),as well as the negative control (experiment 1). Gene BDO OD expressedConditions (μM) (600 nm) BDO/OD none Aerobic 0 13.4 0 none Microaerobic0.5 6.7 0.09 none Anaerobic 2.2 1.26 1.75 0002 Aerobic 138.3 9.12 15.20002 Microaerobic 48.2 5.52 8.73 0002 Anaerobic 54.7 1.35 40.5 0008nAerobic 255.8 5.37 47.6 0008n Microaerobic 127.9 3.05 41.9 0008nAnaerobic 60.8 0.62 98.1 0035 Aerobic 21.3 14.0 1.52 0035 Microaerobic13.1 4.14 3.16 0035 Anaerobic 21.3 1.06 20.1

As discussed above, it may be advantageous to use a route for converting4-HB to 4-HB-CoA that does not generate acetate as a byproduct. To thisaim, we tested the use of phosphotransbutyrylase (ptb) and butyratekinase (bk) from C. acetobutylicum to carry out this conversion viasteps 10 and 11 in FIG. 1. The native ptb/bk operon from C.acetobutylicum (genes 0020 and 0021) was cloned and expressed in pZA33.Extracts from cells containing the resulting construct were taken andassayed for the two enzyme activities as described herein. The specificactivity of BK was approximately 65 U/mg, while the specific activity ofPTB was approximately 5 U/mg. One unit (U) of activity is defined asconversion of 1 μM substrate in 1 minute at room temperature. Finally,the construct was tested for participation in the conversion of 4-HB toBDO. Host strains were transformed with the pZA33-0020-0021 constructdescribed and pZE13-0002, and compared to use of cat2 in BDO productionusing the aerobic procedure used above in FIG. 5. The BK/PTB strainproduced 1 mM BDO, compared to 2 mM when using cat2 (Table 11).Interestingly, the results were dependent on whether the host straincontained a deletion in the native adhE gene.

TABLE 11 Absolute and normalized BDO concentrations from cultures ofcells expressing adhE2 from C. acetobutylicum in pZE13 along with eithercat2 from P. gingivalis (0034) or the PTB/BK genes from C.acetobutylicum on pZA33. Host strains were either MG1655 lacI^(Q) orMG1655 ΔadhE lacI^(Q). BDO OD Genes Host Strain (μM) (600 nm) BDO/OD0034 MG1655 lacI^(Q) 0.827 19.9 0.042 0020 + 0021 MG1655 lacI^(Q) 0.0079.8 0.0007 0034 MG1655 ΔadhE 2.084 12.5 0.166 lacI^(Q) 0020 + 0021MG1655 ΔadhE 0.975 18.8 0.052 lacI^(Q)

Production of BDO from Glucose. The final step of pathway corroborationis to express both the 4-HB and BDO segments of the pathway in E. coliand demonstrate production of BDO in glucose minimal medium. Newplasmids were constructed so that all the required genes fit on twoplasmids. In general, cat1, adhE, and sucD genes were expressed frompZE13, and cat2 and 4-HBd were expressed from pZA33. Variouscombinations of gene source and gene order were tested in the MG1655lacI^(Q) background. Cells were grown anaerobically in M9 minimal medium(6.78 g/L Na₂HPO₄, 3.0 g/L KH₂PO₄, 0.5 g/L NaCl, 1.0 g/L NH₄Cl, 1 mMMgSO₄, 0.1 mM CaCl₂) supplemented with 20 g/L glucose, 100 mM3-(N-morpholino)propanesulfonic acid (MOPS) to improve the bufferingcapacity, 10 μg/mL thiamine, and the appropriate antibiotics. 0.25 mMIPTG was added approximately 15 hours following inoculation, and culturesupernatant samples taken for BDO, 4-HB, and succinate analysis 24 and48 hours following induction. The production of BDO appeared to show adependency on gene order (Table 12). The highest BDO production, over0.5 mM, was obtained with cat2 expressed first, followed by 4-HBd onpZA33, and cat1 followed by P. gingivalis sucD on pZE13. The addition ofC. acetobutylicum adhE2 in the last position on pZE13 resulted in slightimprovement. 4-HB and succinate were also produced at higherconcentrations.

TABLE 12 Production of BDO, 4-HB, and succinate in recombinant E. colistrains expressing combinations of BDO pathway genes, grown in minimalmedium supplemented with 20 g/L glucose. Concentrations are given in mM.24 Hours 48 Hours Induction OD600 OD600 Sample pZE13 pZA33 OD nm Su 4HBBDO nm Su 4HB BDO 1 cat1(0004)-sucD(0035) 4hbd (0036)-cat2(0034) 0.921.29 5.44 1.37 0.240 1.24 6.42 1.49 0.280 2 cat1(0004)-sucD(0008N) 4hbd(0036)-cat2(0034) 0.36 1.11 6.90 1.24 0.011 1.06 7.63 1.33 0.011 3adhE(0002)-cat1(0004)-sucD(0035) 4hbd (0036)-cat2(0034) 0.20 0.44 0.341.84 0.050 0.60 1.93 2.67 0.119 4 cat1(0004)-sucD(0035)-adhE(0002) 4hbd(0036)-cat2(0034) 1.31 1.90 9.02 0.73 0.073 1.95 9.73 0.82 0.077 5adhE(0002)-cat1(0004)-sucD(0008N) 4hbd (0036)-cat2(0034) 0.17 0.45 1.041.04 0.008 0.94 7.13 1.02 0.017 6 cat1(0004)-sucD(0008N)-adhE(0002) 4hbd(0036)-cat2(0034) 1.30 1.77 10.47 0.25 0.004 1.80 11.49 0.28 0.003 7cat1(0004)-sucD(0035) cat2(0034)-4hbd(0036) 1.09 1.29 5.63 2.15 0.4611.38 6.66 2.30 0.520 8 cat1(0004)-sucD(0008N) cat2(0034)-4hbd(0036) 1.812.01 11.28 0.02 0.000 2.24 11.13 0.02 0.000 9adhE(0002)-cat1(0004)-sucD(0035) cat2(0034)-4hbd(0036) 0.24 1.99 2.022.32 0.106 0.89 4.85 2.41 0.186 10 cat1(0004)-sucD(0035)-adhE(0002)cat2(0034)-4hbd(0036) 0.98 1.17 5.30 2.08 0.569 1.33 6.15 2.14 0.640 11adhE(0002)-cat1(0004)-sucD(0008N) cat2(0034)-4hbd(0036) 0.20 0.53 1.382.30 0.019 0.91 8.10 1.49 0.034 12 cat1(0004)-sucD(0008N)-adhE(0002)cat2(0034)-4hbd(0036) 2.14 2.73 12.07 0.16 0.000 3.10 11.79 0.17 0.00213 vector only vector only 2.11 2.62 9.03 0.01 0.000 3.00 12.05 0.010.000

Analysis of BDO, 4-HB and succinate by GCMS. BDO, 4-HB and succinate infermentation and cell culture samples were derivatized by silylation andquantitatively analyzed by GCMS using methods adapted from literaturereports ((Simonov et al., J. Anal Chem. 59:965-971 (2004)). Thedeveloped method demonstrated good sensitivity down to 1 μM, linearityup to at least 25 mM, as well as excellent selectivity andreproducibility.

Sample preparation was performed as follows: 100 μL filtered (0.2 μm or0.45 μm syringe filters) samples, e.g. fermentation broth, cell cultureor standard solutions, were dried down in a Speed Vac Concentrator(Savant SVC-100H) for approximately 1 hour at ambient temperature,followed by the addition of 20 μL 10 mM cyclohexanol solution, as aninternal standard, in dimethylformamide. The mixtures were vortexed andsonicated in a water bath (Branson 3510) for 15 min to ensurehomogeneity. 100 μL silylation derivatization reagent, N,O-bis(trimethylsilyl)trifluoro-acetimide (BSTFA) with 1%trimethylchlorosilane, was added, and the mixture was incubated at 70°C. for 30 min. The derivatized samples were centrifuged for 5 min, andthe clear solutions were directly injected into GCMS. All the chemicalsand reagents were from Sigma-Aldrich, with the exception of BDO whichwas purchased from J. T. Baker.

GCMS was performed on an Agilent gas chromatograph 6890N, interfaced toa mass-selective detector (MSD) 5973N operated in electron impactionization (E1) mode has been used for the analysis. A DB-5MS capillarycolumn (J&W Scientific, Agilent Technologies), 30m×0.25 mm i.d.×0.25 μmfilm thickness, was used. The GC was operated in a split injection modeintroducing 1 μL of sample at 20:1 split ratio. The injection porttemperature was 250° C. Helium was used as a carrier gas, and the flowrate was maintained at 1.0 mL/min. A temperature gradient program wasoptimized to ensure good resolution of the analytes of interest andminimum matrix interference. The oven was initially held at 80° C. for 1min, then ramped to 120° C. at 2° C./min, followed by fast ramping to320° C. at 100° C./min and final hold for 6 min at 320° C. The MSinterface transfer line was maintained at 280° C. The data were acquiredusing ‘lowmass’ MS tune settings and 30-400 m/z mass-range scan. Thetotal analysis time was 29 min including 3 min solvent delay. Theretention times corresponded to 5.2, 10.5, 14.0 and 18.2 min forBSTFA-derivatized cyclohexanol, BDO, 4-HB and succinate, respectively.For quantitative analysis, the following specific mass fragments wereselected (extracted ion chromatograms): m/z 157 for internal standardcyclohexanol, 116 for BDO, and 147 for both 4-HB and succinate. Standardcalibration curves were constructed using analyte solutions in thecorresponding cell culture or fermentation medium to match sample matrixas close as possible. GCMS data were processed using Environmental DataAnalysis ChemStation software (Agilent Technologies).

The results indicated that most of the 4-HB and BDO produced werelabeled with ¹³C (FIG. 6, right-hand sides). Mass spectra from aparallel culture grown in unlabeled glucose are shown for comparison(FIG. 6, left-hand sides). Note that the peaks seen are for fragments ofthe derivatized molecule containing different numbers of carbon atomsfrom the metabolite. The derivatization reagent also contributes somecarbon and silicon atoms that naturally-occurring label distribution, sothe results are not strictly quantitative.

Production of BDO from 4-HB using alternate pathways. The variousalternate pathways were also tested for BDO production. This includesuse of the native E. coli SucCD enzyme to convert succinate tosuccinyl-CoA (Table 13, rows 2-3), use of α-ketoglutarate decarboxylasein the α-ketoglutarate pathway (Table 13, row 4), and use of PTB/BK asan alternate means to generate the CoA-derivative of 4HB (Table 13, row1). Strains were constructed containing plasmids expressing the genesindicated in Table 13, which encompass these variants. The results showthat in all cases, production of 4-HB and BDO occurred (Table 13).

TABLE 13 Production of BDO, 4-HB, and succinate in recombinant E. colistrains genes for different BDO pathway variants, grown anaerobically inminimal medium supplemented with 20 g/L glucose, and harvested 24 hoursafter induction with 0.1 mM IPTG. Concentrations are given in mM. Geneson pZE13 Genes on pZA33 Succinate 4-HB BDO 0002 + 0004 + 00350020n-0021n-0036 0.336 2.91 0.230 0038 + 0035 0034-0036 0.814 2.81 0.1260038 + 0035 0036-0034 0.741 2.57 0.114 0035 + 0032 0034-0036 5.01 0.5380.154

EXAMPLE III Biosynthesis of 4-Hydroxybutanoic Acid, γ-Butyrolactone and1,4-Butanediol

This Example describes the biosynthetic production of 4-hydroxybutanoicacid, γ-butyrolactone and 1,4-butanediol using fermentation and otherbioprocesses.

Methods for the integration of the 4-HB fermentation step into acomplete process for the production of purified GBL, 1,4-butanediol(BDO) and tetrahydrofuran (THF) are described below. Since 4-HB and GBLare in equilibrium, the fermentation broth will contain both compounds.At low pH this equilibrium is shifted to favor GBL. Therefore, thefermentation can operate at pH 7.5 or less, generally pH 5.5 or less.After removal of biomass, the product stream enters into a separationstep in which GBL is removed and the remaining stream enriched in 4-HBis recycled. Finally, GBL is distilled to remove any impurities. Theprocess operates in one of three ways: 1) fed-batch fermentation andbatch separation; 2) fed-batch fermentation and continuous separation;3) continuous fermentation and continuous separation. The first two ofthese modes are shown schematically in FIG. 7. The integratedfermentation procedures described below also are used for the BDOproducing cells of the invention for biosynthesis of BDO and subsequentBDO family products.

Fermentation protocol to produce 4-HB/GBL (batch): The productionorganism is grown in a 10 L bioreactor sparged with an N₂/CO₂ mixture,using 5 L broth containing 5 g/L potassium phosphate, 2.5 g/L ammoniumchloride, 0.5 g/L magnesium sulfate, and 30 g/L corn steep liquor, andan initial glucose concentration of 20 g/L. As the cells grow andutilize the glucose, additional 70% glucose is fed into the bioreactorat a rate approximately balancing glucose consumption. The temperatureof the bioreactor is maintained at 30 degrees C. Growth continues forapproximately 24 hours, until 4-HB reaches a concentration of between20-200 g/L, with the cell density being between 5 and 10 g/L. The pH isnot controlled, and will typically decrease to pH 3-6 by the end of therun. Upon completion of the cultivation period, the fermenter contentsare passed through a cell separation unit (e.g., centrifuge) to removecells and cell debris, and the fermentation broth is transferred to aproduct separations unit. Isolation of 4-HB and/or GBL would take placeby standard separations procedures employed in the art to separateorganic products from dilute aqueous solutions, such as liquid-liquidextraction using a water immiscible organic solvent (e.g., toluene) toprovide an organic solution of 4-HB/GBL. The resulting solution is thensubjected to standard distillation methods to remove and recycle theorganic solvent and to provide GBL (boiling point 204-205° C.) which isisolated as a purified liquid.

Fermentation protocol to produce 4-HB/GBL (fully continuous): Theproduction organism is first grown up in batch mode using the apparatusand medium composition described above, except that the initial glucoseconcentration is 30-50 g/L. When glucose is exhausted, feed medium ofthe same composition is supplied continuously at a rate between 0.5 L/hrand 1 L/hr, and liquid is withdrawn at the same rate. The 4-HBconcentration in the bioreactor remains constant at 30-40 g/L, and thecell density remains constant between 3-5 g/L. Temperature is maintainedat 30 degrees C., and the pH is maintained at 4.5 using concentratedNaOH and HCl, as required. The bioreactor is operated continuously forone month, with samples taken every day to assure consistency of 4-HBconcentration. In continuous mode, fermenter contents are constantlyremoved as new feed medium is supplied. The exit stream, containingcells, medium, and products 4-HB and/or GBL, is then subjected to acontinuous product separations procedure, with or without removing cellsand cell debris, and would take place by standard continuous separationsmethods employed in the art to separate organic products from diluteaqueous solutions, such as continuous liquid-liquid extraction using awater immiscible organic solvent (e.g., toluene) to provide an organicsolution of 4-HB/GBL. The resulting solution is subsequently subjectedto standard continuous distillation methods to remove and recycle theorganic solvent and to provide GBL (boiling point 204-205° C.) which isisolated as a purified liquid.

GBL Reduction Protocol: Once GBL is isolated and purified as describedabove, it will then be subjected to reduction protocols such as thosewell known in the art (references cited) to produce 1,4-butanediol ortetrahydrofuran (THF) or a mixture thereof. Heterogeneous or homogeneoushydrogenation catalysts combined with GBL under hydrogen pressure arewell known to provide the products 1,4-butanediol or tetrahydrofuran(THF) or a mixture thereof. It is important to note that the 4-HB/GBLproduct mixture that is separated from the fermentation broth, asdescribed above, may be subjected directly, prior to GBL isolation andpurification, to these same reduction protocols to provide the products1,4-butanediol or tetrahydrofuran or a mixture thereof. The resultingproducts, 1,4-butanediol and THF are then isolated and purified byprocedures well known in the art.

Fermentation and hydrogenation protocol to produce BDO or THF directly(batch):

Cells are grown in a 10 L bioreactor sparged with an N₂/CO₂ mixture,using 5 L broth containing 5 g/L potassium phosphate, 2.5 g/L ammoniumchloride, 0.5 g/L magnesium sulfate, and 30 g/L corn steep liquor, andan initial glucose concentration of 20 g/L. As the cells grow andutilize the glucose, additional 70% glucose is fed into the bioreactorat a rate approximately balancing glucose consumption. The temperatureof the bioreactor is maintained at 30 degrees C. Growth continues forapproximately 24 hours, until 4-HB reaches a concentration of between20-200 g/L, with the cell density being between 5 and 10 g/L. The pH isnot controlled, and will typically decrease to pH 3-6 by the end of therun. Upon completion of the cultivation period, the fermenter contentsare passed through a cell separation unit (e.g., centrifuge) to removecells and cell debris, and the fermentation broth is transferred to areduction unit (e.g., hydrogenation vessel), where the mixture 4-HB/GBLis directly reduced to either 1,4-butanediol or THF or a mixturethereof. Following completion of the reduction procedure, the reactorcontents are transferred to a product separations unit. Isolation of1,4-butanediol and/or THF would take place by standard separationsprocedures employed in the art to separate organic products from diluteaqueous solutions, such as liquid-liquid extraction using a waterimmiscible organic solvent (e.g., toluene) to provide an organicsolution of 1,4-butanediol and/or THF. The resulting solution is thensubjected to standard distillation methods to remove and recycle theorganic solvent and to provide 1,4-butanediol and/or THF which areisolated as a purified liquids.

Fermentation and hydrogenation protocol to produce BDO or THF directly(fully continuous): The cells are first grown up in batch mode using theapparatus and medium composition described above, except that theinitial glucose concentration is 30-50 g/L. When glucose is exhausted,feed medium of the same composition is supplied continuously at a ratebetween 0.5 L/hr and 1 L/hr, and liquid is withdrawn at the same rate.The 4-HB concentration in the bioreactor remains constant at 30-40 g/L,and the cell density remains constant between 3-5 g/L. Temperature ismaintained at 30 degrees C., and the pH is maintained at 4.5 usingconcentrated NaOH and HCl, as required. The bioreactor is operatedcontinuously for one month, with samples taken every day to assureconsistency of 4-HB concentration. In continuous mode, fermentercontents are constantly removed as new feed medium is supplied. The exitstream, containing cells, medium, and products 4-HB and/or GBL, is thenpassed through a cell separation unit (e.g., centrifuge) to remove cellsand cell debris, and the fermentation broth is transferred to acontinuous reduction unit (e.g., hydrogenation vessel), where themixture 4-HB/GBL is directly reduced to either 1,4-butanediol or THF ora mixture thereof. Following completion of the reduction procedure, thereactor contents are transferred to a continuous product separationsunit. Isolation of 1,4-butanediol and/or THF would take place bystandard continuous separations procedures employed in the art toseparate organic products from dilute aqueous solutions, such asliquid-liquid extraction using a water immiscible organic solvent (e.g.,toluene) to provide an organic solution of 1,4-butanediol and/or THF.The resulting solution is then subjected to standard continuousdistillation methods to remove and recycle the organic solvent and toprovide 1,4-butanediol and/or THF which are isolated as a purifiedliquids.

Fermentation protocol to produce BDO directly (batch): The productionorganism is grown in a 10 L bioreactor sparged with an N₂/CO₂ mixture,using 5 L broth containing 5 g/L potassium phosphate, 2.5 g/L ammoniumchloride, 0.5 g/L magnesium sulfate, and 30 g/L corn steep liquor, andan initial glucose concentration of 20 g/L. As the cells grow andutilize the glucose, additional 70% glucose is fed into the bioreactorat a rate approximately balancing glucose consumption. The temperatureof the bioreactor is maintained at 30 degrees C. Growth continues forapproximately 24 hours, until BDO reaches a concentration of between20-200 g/L, with the cell density generally being between 5 and 10 g/L.Upon completion of the cultivation period, the fermenter contents arepassed through a cell separation unit (e.g., centrifuge) to remove cellsand cell debris, and the fermentation broth is transferred to a productseparations unit. Isolation of BDO would take place by standardseparations procedures employed in the art to separate organic productsfrom dilute aqueous solutions, such as liquid-liquid extraction using awater immiscible organic solvent (e.g., toluene) to provide an organicsolution of BDO. The resulting solution is then subjected to standarddistillation methods to remove and recycle the organic solvent and toprovide BDO (boiling point 228-229° C.) which is isolated as a purifiedliquid.

Fermentation protocol to produce BDO directly (fully continuous): Theproduction organism is first grown up in batch mode using the apparatusand medium composition described above, except that the initial glucoseconcentration is 30-50 g/L. When glucose is exhausted, feed medium ofthe same composition is supplied continuously at a rate between 0.5 L/hrand 1 L/hr, and liquid is withdrawn at the same rate. The BDOconcentration in the bioreactor remains constant at 30-40 g/L, and thecell density remains constant between 3-5 g/L. Temperature is maintainedat 30 degrees C., and the pH is maintained at 4.5 using concentratedNaOH and HCl, as required. The bioreactor is operated continuously forone month, with samples taken every day to assure consistency of BDOconcentration. In continuous mode, fermenter contents are constantlyremoved as new feed medium is supplied. The exit stream, containingcells, medium, and the product BDO, is then subjected to a continuousproduct separations procedure, with or without removing cells and celldebris, and would take place by standard continuous separations methodsemployed in the art to separate organic products from dilute aqueoussolutions, such as continuous liquid-liquid extraction using a waterimmiscible organic solvent (e.g., toluene) to provide an organicsolution of BDO. The resulting solution is subsequently subjected tostandard continuous distillation methods to remove and recycle theorganic solvent and to provide BDO (boiling point 228-229° C.) which isisolated as a purified liquid (mpt 20° C.).

EXAMPLE IV Exemplary BDO Pathways

This example describes exemplary enzymes and corresponding genes for1,4-butandiol (BDO) synthetic pathways.

Exemplary BDO synthetic pathways are shown in FIGS. 8-13. The pathwaysdepicted in FIGS. 8-13 are from common central metabolic intermediatesto 1,4-butanediol. All transformations depicted in FIGS. 8-13 fall intothe 18 general categories of transformations shown in Table 14. Below isdescribed a number of biochemically characterized candidate genes ineach category. Specifically listed are genes that can be applied tocatalyze the appropriate transformations in FIGS. 9-13 when cloned andexpressed in a host organism. The top three exemplary genes for each ofthe key steps in FIGS. 9-13 are provided in Tables 15-23 (see below).Exemplary genes were provided for the pathways depicted in FIG. 8 aredescribed herein.

TABLE 14 Enzyme types required to convert common central metabolicintermediates into 1,4-butanediol. The first three digits of each labelcorrespond to the first three Enzyme Commission number digits whichdenote the general type of transformation independent of substratespecificity. Label Function 1.1.1.a Oxidoreductase (ketone to hydroxylor aldehyde to alcohol) 1.1.1.c Oxidoreductase (2 step, acyl-CoA toalcohol) 1.2.1.b Oxidoreductase (acyl-CoA to aldehyde) 1.2.1.cOxidoreductase (2-oxo acid to acyl-CoA, decarboxylation) 1.2.1.dOxidoreductase (phosphorylating/dephosphorylating) 1.3.1.aOxidoreductase operating on CH—CH donors 1.4.1.a Oxidoreductaseoperating on amino acids 2.3.1.a Acyltransferase (transferring phosphategroup) 2.6.1.a Aminotransferase 2.7.2.a Phosphotransferase, carboxylgroup acceptor 2.8.3.a Coenzyme-A transferase 3.1.2.a Thiolesterhydrolase (CoA specific) 4.1.1.a Carboxy-lyase 4.2.1.a Hydro-lyase4.3.1.a Ammonia-lyase 5.3.3.a Isomerase 5.4.3.a Aminomutase 6.2.1.aAcid-thiol ligase1.1.1.a-Oxidoreductase (Aldehyde to Alcohol or Ketone to Hydroxyl)

Aldehyde to Alcohol. Exemplary genes encoding enzymes that catalyze theconversion of an aldehyde to alcohol, that is, alcohol dehydrogenase orequivalently aldehyde reductase, include alrA encoding a medium-chainalcohol dehydrogenase for C2-C14 (Tani et al. Appl. Environ. Microbiol.66:5231-5235 (2000)), ADH2 from Saccharomyces cerevisiae (Atsumi et al.Nature 451:86-89 (2008)), yqhD from E. coli which has preference formolecules longer than C(3) (Sulzenbacher et al. Journal of MolecularBiology 342:489-502 (2004)), and bdh I and bdh II from C. acetobutylicumwhich converts butyryaldehyde into butanol (Walter et al. Journal ofBacteriology 174:7149-7158 (1992)). The protein sequences for each ofthese exemplary gene products, if available, can be found using thefollowing GenBank accession numbers:

Gene Accession No. GI No. Organism alrA BAB12273.1 9967138 Acinetobactersp. Strain M-1 ADH2 NP_014032.1 6323961 Saccharymyces cerevisiae yqhDNP_417484.1 16130909 Escherichia coli bdh I NP_349892.1 15896543Clostridium acetobutylicum bdh II NP_349891.1 15896542 Clostridiumacetobutylicum

Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity (EC1.1.1.61) also fall into this category. Such enzymes have beencharacterized in Ralstonia eutropha (Bravo et al. J. Forensic Sci.49:379-387 (2004), Clostridium kluyveri (Wolff et al. Protein Expr.Purif. 6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz et al. J.Biol. Chem. 278:41552-41556 (2003)).

Gene Accession No. GI No. Organism 4hbd YP_726053.1 113867564 Ralstoniaeutropha H16 4hbd L21902.1 146348486 Clostridium kluyveri DSM 555 4hbdQ94B07 75249805 Arabidopsis thaliana

Another exemplary enzyme is 3-hydroxyisobutyrate dehydrogenase whichcatalyzes the reversible oxidation of 3-hydroxyisobutyrate tomethylmalonate semialdehyde. This enzyme participates in valine, leucineand isoleucine degradation and has been identified in bacteria,eukaryotes, and mammals. The enzyme encoded by P84067 from Thermusthermophilus HB8 has been structurally characterized (Lokanath et al. JMol Biol 352:905-17 (2005)). The reversibility of the human3-hydroxyisobutyrate dehydrogenase was demonstrated usingisotopically-labeled substrate (Manning et al. Biochem J 231:481-484(1985)). Additional genes encoding this enzyme include 3hidh in Homosapiens (Hawes et al. Methods Enzymol. 324:218-228 (2000)) andOryctolagus cuniculus (Chowdhury et al. Biosci. Biotechnol Biochem.60:2043-2047 (1996); Hawes et al. Methods Enzymol. 324:218-228 (2000)),mmsb in Pseudomonas aeruginosa, and dhat in Pseudomonas putida (Aberhartet al. J Chem.Soc. [Perkin 1] 6:1404-1406 (1979); Chowdhury et al.Biosci. Biotechnol Biochem. 67:438-441 (2003); Chowdhury et al. Biosci.Biotechnol Biochem. 60:2043-2047 (1996)).

Gene Accession No. GI No. Organism P84067 P84067 75345323 Thermusthermophilus mmsb P28811.1 127211 Pseudomonas aeruginosa dhat Q59477.12842618 Pseudomonas putida 3hidh P31937.2 12643395 Homo sapiens 3hidhP32185.1 416872 Oryctolagus cuniculus

Several 3-hydroxyisobutyrate dehydrogenase enzymes have also been shownto convert malonic semialdehyde to 3-hydroxyproprionic acid (3-HP).Three gene candidates exhibiting this activity are mmsB from Pseudomonasaeruginosa PAO1(62), mmsB from Pseudomonas putida KT2440 (Liao et al.,US Publication 2005/0221466) and mmsB from Pseudomonas putida E23(Chowdhury et al., Biosci. Biotechnol. Biochem. 60:2043-2047 (1996)). Anenzyme with 3-hydroxybutyrate dehydrogenase activity in Alcaligenesfaecalis M3A has also been identified (Gokam et al., U.S. Pat. No.7,393,676; Liao et al., US Publication No. 2005/0221466). Additionalgene candidates from other organisms including Rhodobacter spaeroidescan be inferred by sequence similarity.

Gene Accession No. GI No. Organism mmsB AAA25892.1 151363 Pseudomonasaeruginosa mmsB NP_252259.1 15598765 Pseudomonas aeruginosa PAO1 mmsBNP_746775.1 26991350 Pseudomonas putida KT2440 mmsB JC7926 60729613Pseudomonas putida E23 orfB1 AAL26884 16588720 Rhodobacter spaeroides

The conversion of malonic semialdehyde to 3-HP can also be accomplishedby two other enzymes: NADH-dependent 3-hydroxypropionate dehydrogenaseand NADPH-dependent malonate semialdehyde reductase. An NADH-dependent3-hydroxypropionate dehydrogenase is thought to participate inbeta-alanine biosynthesis pathways from propionate in bacteria andplants (Rathinasabapathi, B. Journal of Plant Pathology 159:671-674(2002); Stadtman, E. R. J. Am. Chem. Soc. 77:5765-5766 (1955)). Thisenzyme has not been associated with a gene in any organism to date.NADPH-dependent malonate semialdehyde reductase catalyzes the reversereaction in autotrophic CO₂-fixing bacteria. Although the enzymeactivity has been detected in Metallosphaera sedula, the identity of thegene is not known (Alber et al. J. Bacteriol. 188:8551-8559 (2006)).

Ketone to Hydroxyl. There exist several exemplary alcohol dehydrogenasesthat convert a ketone to a hydroxyl functional group. Two such enzymesfrom E. coli are encoded by malate dehydrogenase (mdh) and lactatedehydrogenase (ldhA). In addition, lactate dehydrogenase from Ralstoniaeutropha has been shown to demonstrate high activities on substrates ofvarious chain lengths such as lactate, 2-oxobutyrate, 2-oxopentanoateand 2-oxoglutarate (Steinbuchel and. Schlegel, Eur. J. Biochem.130:329-334 (1983)). Conversion of alpha-ketoadipate intoalpha-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, anenzyme reported to be found in rat and in human placenta (Suda et al.Arch. Biochem. Biophys. 176:610-620 (1976); Suda et al. Biochem.Biophys. Res. Commun. 77:586-591 (1977)). An additional candidate forthis step is the mitochondrial 3-hydroxybutyrate dehydrogenase (bdh)from the human heart which has been cloned and characterized (Marks etal. J. Biol. Chem. 267:15459-15463 (1992)). This enzyme is adehydrogenase that operates on a 3-hydroxyacid. Another exemplaryalcohol dehydrogenase converts acetone to isopropanol as was shown in C.beijerinckii (Ismaiel et al. J. Bacteriol. 175:5097-5105 (1993)) and T.brockii (Lamed et al. Biochem. J. 195:183-190 (1981); Peretz andBurstein Biochemistry 28:6549-6555 (1989)).

Gene Accession No. GI No. Organism mdh AAC76268.1 1789632 Escherichiacoli ldhA NP_415898.1 16129341 Escherichia coli ldh YP_725182.1113866693 Ralstonia eutropha bdh AAA58352.1 177198 Homo sapiens adhAAA23199.2 60592974 Clostridium beijerinckii NRRL B593 adh P14941.1113443 Thermoanaerobacter brockii HTD4

Exemplary 3-hydroxyacyl dehydrogenases which convert acetoacetyl-CoA to3-hydroxybutyryl-CoA include hbd from C. acetobutylicum (Boynton et al.Journal of Bacteriology 178:3015-3024 (1996)), hbd from C. beijerinckii(Colby et al. Appl Environ. Microbiol 58:3297-3302 (1992)), and a numberof similar enzymes from Metallosphaera sedula (Berg et al. Archaea.Science 318:1782-1786 (2007)).

Gene Accession No. GI No. Organism hbd NP_349314.1 15895965 Clostridiumacetobutylicum hbd AAM14586.1 20162442 Clostridium beijerinckiiMsed_1423 YP_001191505 146304189 Metallosphaera sedula Msed_0399YP_001190500 146303184 Metallosphaera sedula Msed_0389 YP_001190490146303174 Metallosphaera sedula Msed_1993 YP_001192057 146304741Metallosphaera sedula1.1.1.c-Oxidoredutase (2 Step, Acyl-CoA to Alcohol)

Exemplary 2-step oxidoreductases that convert an acyl-CoA to alcoholinclude those that transform substrates such as acetyl-CoA to ethanol(for example, adhE from E. coli (Kessler et al. FEBS. Lett. 281:59-63(1991)) and butyryl-CoA to butanol (for example, adhE2 from C.acetobutylicum (Fontaine et al. J. Bacteriol. 184:821-830 (2002)). Inaddition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhEin Leuconostoc mesenteroides has been shown to oxide the branched chaincompound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al. J. Gen.Appl. Microbiol. 18:43-55 (1972); Koo et al. Biotechnol Lett. 27:505-510(2005)).

Gene Accession No. GI No. Organism adhE NP_415757.1 16129202 Escherichiacoli adhE2 AAK09379.1 12958626 Clostridium acetobutylicum adhEAAV66076.1 55818563 Leuconostoc mesenteroides

Another exemplary enzyme can convert malonyl-CoA to 3-HP. AnNADPH-dependent enzyme with this activity has characterized inChloroflexus aurantiacus where it participates in the3-hydroxypropionate cycle (Hugler et al., J. Bacteriol. 184:2404-2410(2002); Strauss and Fuchs, Eur. J. Biochem. 215:633-643 (1993)). Thisenzyme, with a mass of 300 kDa, is highly substrate-specific and showslittle sequence similarity to other known oxidoreductases (Hugler etal., J. Bacteriol. 184:2404-2410 (2002)). No enzymes in other organismshave been shown to catalyze this specific reaction; however there isbioinformatic evidence that other organisms may have similar pathways(Klatt et al., Environ. Microbiol. 9:2067-2078 (2007)). Enzymecandidates in other organisms including Roseiflexus castenholzii,Erythrobacter sp. NAP1 and marine gamma proteobacterium HTCC2080 can beinferred by sequence similarity.

Gene Accession No. GI No. Organism mcr AAS20429.1 42561982 Chloroflexusaurantiacus Rcas_2929 YP_001433009.1 156742880 Roseiflexus castenholziiNAP1_02720 ZP_01039179.1 85708113 Erythrobacter sp. NAP1 MGP2080_00535ZP_01626393.1 119504313 marine gamma proteobacterium HTCC2080

Longer chain acyl-CoA molecules can be reduced by enzymes such as thejojoba (Simmondsia chinensis) FAR which encodes an alcohol-forming fattyacyl-CoA reductase. Its overexpression in E. coli resulted in FARactivity and the accumulation of fatty alcohol (Metz et al. PlantPhysiology 122:635-644) 2000)).

Gene Accession No. GI No. Organism FAR AAD38039.1 5020215 Simmondsiachinensis1.2.1.b-Oxidoreductase (Acyl-CoA to Aldehyde)

Several acyl-CoA dehydrogenases are capable of reducing an acyl-CoA toits corresponding aldehyde. Exemplary genes that encode such enzymesinclude the Acinetobacter calcoaceticus acr1 encoding a fatty acyl-CoAreductase (Reiser and Somerville, J. Bacteriology 179:2969-2975 (1997)),the Acinetobacter sp. M-1 fatty acyl-CoA reductase (Ishige et al. Appl.Environ. Microbiol. 68:1192-1195 (2002)), and a CoA- and NADP-dependentsuccinate semialdehyde dehydrogenase encoded by the sucD gene inClostridium kluyveri (Sohling and Gottschalk J Bacteriol 178:871-80(1996); Sohling and Gottschalk J. Bacteriol. 178:871-880 (1996)). SucDof P. gingivalis is another succinate semialdehyde dehydrogenase(Takahashi et al. J. Bacteriol. 182:4704-4710 (2000)). The enzymeacylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG,is yet another as it has been demonstrated to oxidize and acylateacetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde andformaldehyde (Powlowski et al. J Bacteriol. 175:377-385 (1993)).

Gene Accession No. GI No. Organism acr1 YP_047869.1 50086359Acinetobacter calcoaceticus acr1 AAC45217 1684886 Acinetobacter baylyiacr1 BAB85476.1 18857901 Acinetobacter sp. Strain M-1 sucD P38947.1730847 Clostridium kluyveri sucD NP_904963.1 34540484 Porphyromonasgingivalis bphG BAA03892.1 425213 Pseudomonas sp

An additional enzyme type that converts an acyl-CoA to its correspondingaldehyde is malonyl-CoA reductase which transforms malonyl-CoA tomalonic semialdehyde. Malonyl-CoA reductase is a key enzyme inautotrophic carbon fixation via the 3-hydroxypropionate cycle inthermoacidophilic archael bacteria (Berg et al. Science 318:1782-1786(2007); Thauer, R. K. Science 318:1732-1733 (2007)). The enzyme utilizesNADPH as a cofactor and has been characterized in Metallosphaera andSulfolobus spp (Alber et al. J. Bacteriol. 188:8551-8559 (2006); Hugleret al. J. Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded byMsed_0709 in Metallosphaera sedula (Alber et al. J. Bacteriol.188:8551-8559 (2006); Berg et al. Science 318:1782-1786 (2007)). A geneencoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned andheterologously expressed in E. coli (Alber et al. J. Bacteriol.188:8551-8559 (2006)). Although the aldehyde dehydrogenase functionalityof these enzymes is similar to the bifunctional dehydrogenase fromChloroflexus aurantiacus, there is little sequence similarity. Bothmalonyl-CoA reductase enzyme candidates have high sequence similarity toaspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reductionand concurrent dephosphorylation of aspartyl-4-phosphate to aspartatesemialdehyde. Additional gene candidates can be found by sequencehomology to proteins in other organisms including Sulfolobussolfataricus and Sulfolobus acidocaldarius.

Gene Accession No. GI No. Organism Msed_0709 YP_001190808.1 146303492Metallosphaera sedula mcr NP_378167.1 15922498 Sulfolobus tokodaii asd-2NP_343563.1 15898958 Sulfolobus solfataricus Saci_2370 YP_256941.170608071 Sulfolobus acidocaldarius1.2.1.c-Oxidoreductase (2-Oxo Acid to Acyl-CoA, Decarboxylation)

Enzymes in this family include 1) branched-chain 2-keto-aciddehydrogenase, 2) alpha-ketoglutarate dehydrogenase, and 3) the pyruvatedehydrogenase multienzyme complex (PDHC). These enzymes are multi-enzymecomplexes that catalyze a series of partial reactions which result inacylating oxidative decarboxylation of 2-keto-acids. Each of the2-keto-acid dehydrogenase complexes occupies key positions inintermediary metabolism, and enzyme activity is typically tightlyregulated (Fries et al. Biochemistry 42:6996-7002 (2003)). The enzymesshare a complex but common structure composed of multiple copies ofthree catalytic components: alpha-ketoacid decarboxylase (E1),dihydrolipoamide acyltransferase (E2) and dihydrolipoamide dehydrogenase(E3). The E3 component is shared among all 2-keto-acid dehydrogenasecomplexes in an organism, while the E1 and E2 components are encoded bydifferent genes. The enzyme components are present in numerous copies inthe complex and utilize multiple cofactors to catalyze a directedsequence of reactions via substrate channeling. The overall size ofthese dehydrogenase complexes is very large, with molecular massesbetween 4 and 10 million Da (that is, larger than a ribosome).

Activity of enzymes in the 2-keto-acid dehydrogenase family is normallylow or limited under anaerobic conditions in E. coli. Increasedproduction of NADH (or NADPH) could lead to a redox-imbalance, and NADHitself serves as an inhibitor to enzyme function. Engineering effortshave increased the anaerobic activity of the E. coli pyruvatedehydrogenase complex (Kim et al. Appl. Environ. Microbiol. 73:1766-1771(2007); Kim et al. J. Bacteriol. 190:3851-3858) 2008); Zhou et al.Biotechnol. Lett. 30:335-342 (2008)). For example, the inhibitory effectof NADH can be overcome by engineering an H322Y mutation in the E3component (Kim et al. J. Bacteriol. 190:3851-3858 (2008)). Structuralstudies of individual components and how they work together in complexprovide insight into the catalytic mechanisms and architecture ofenzymes in this family (Aevarsson et al. Nat. Struct. Biol. 6:785-792(1999); Zhou et al. Proc. Natl. Acad. Sci. U.S.A. 98:14802-14807(2001)). The substrate specificity of the dehydrogenase complexes variesin different organisms, but generally branched-chain keto-aciddehydrogenases have the broadest substrate range.

Alpha-ketoglutarate dehydrogenase (AKGD) converts alpha-ketoglutarate tosuccinyl-CoA and is the primary site of control of metabolic fluxthrough the TCA cycle (Hansford, R. G. Curr. Top. Bioenerg. 10:217-278(1980)). Encoded by genes sucA, sucB and lpd in E. coli, AKGD geneexpression is downregulated under anaerobic conditions and during growthon glucose (Park et al. Mol. Microbiol. 15:473-482 (1995)). Although thesubstrate range of AKGD is narrow, structural studies of the catalyticcore of the E2 component pinpoint specific residues responsible forsubstrate specificity (Knapp et al. J. Mol. Biol. 280:655-668 (1998)).The Bacillus subtilis AKGD, encoded by odhAB (E1 and E2) and pdhD (E3,shared domain), is regulated at the transcriptional level and isdependent on the carbon source and growth phase of the organism(Resnekov et al. Mol. Gen. Genet. 234:285-296 (1992)). In yeast, theLPD1 gene encoding the E3 component is regulated at the transcriptionallevel by glucose (Roy and Dawes J. Gen. Microbiol. 133:925-933 (1987)).The E1 component, encoded by KGD1, is also regulated by glucose andactivated by the products of HAP2 and HAP3 (Repetto and Tzagoloff Mol.Cell Biol. 9:2695-2705 (1989)). The AKGD enzyme complex, inhibited byproducts NADH and succinyl-CoA, is well-studied in mammalian systems, asimpaired function of has been linked to several neurological diseases(Tretter and dam-Vizi Philos. Trans. R. Soc. Lond B Biol. Sci.360:2335-2345 (2005)).

Gene Accession No. GI No. Organism sucA NP_415254.1 16128701 Escherichiacoli str. K12 substr. MG1655 sucB NP_415255.1 16128702 Escherichia colistr. K12 substr. MG1655 lpd NP_414658.1 16128109 Escherichia coli str.K12 substr. MG1655 odhA P23129.2 51704265 Bacillus subtilis odhBP16263.1 129041 Bacillus subtilis pdhD P21880.1 118672 Bacillus subtilisKGD1 NP_012141.1 6322066 Saccharomyces cerevisiae KGD2 NP_010432.16320352 Saccharomyces cerevisiae LPD1 NP_116635.1 14318501 Saccharomycescerevisiae

Branched-chain 2-keto-acid dehydrogenase complex (BCKAD), also known as2-oxoisovalerate dehydrogenase, participates in branched-chain aminoacid degradation pathways, converting 2-keto acids derivatives ofvaline, leucine and isoleucine to their acyl-CoA derivatives and CO₂.The complex has been studied in many organisms including Bacillussubtilis (Wang et al. Eur. J. Biochem. 213:1091-1099 (1993)), Rattusnorvegicus (Namba et al. J. Biol. Chem. 244:4437-4447 (1969)) andPseudomonas putida (Sokatch J. Bacteriol. 148:647-652 (1981)). InBacillus subtilis the enzyme is encoded by genes pdhD (E3 component),bfmBB (E2 component), bfmBAA and bfmBAB (E1 component) (Wang et al. Eur.J. Biochem. 213:1091-1099 (1993)). In mammals, the complex is regulatedby phosphorylation by specific phosphatases and protein kinases. Thecomplex has been studied in rat hepatocites (Chicco et al. J. Biol.Chem. 269:19427-19434 (1994)) and is encoded by genes Bckdha (E1 alpha),Bckdhb (E1 beta), Dbt (E2), and Dld (E3). The E1 and E3 components ofthe Pseudomonas putida BCKAD complex have been crystallized (Aevarssonet al. Nat. Struct. Biol. 6:785-792 (1999); Mattevi Science255:1544-1550 (1992)) and the enzyme complex has been studied (Sokatchet al. J. Bacteriol. 148:647-652 (1981)). Transcription of the P. putidaBCKAD genes is activated by the gene product of bkdR (Hester et al. Eur.J. Biochem. 233:828-836 (1995)). In some organisms including Rattusnorvegicus (Paxton et al. Biochem. J. 234:295-303 (1986)) andSaccharomyces cerevisiae (Sinclair et al. Biochem. Mol. Biol. Int.31:911-922 (1993)), this complex has been shown to have a broadsubstrate range that includes linear oxo-acids such as 2-oxobutanoateand alpha-ketoglutarate, in addition to the branched-chain amino acidprecursors. The active site of the bovine BCKAD was engineered to favoralternate substrate acetyl-CoA (Meng and Chuang, Biochemistry33:12879-12885 (1994)).

Gene Accession No. GI No. Organism bfmBB NP_390283.1 16079459 Bacillussubtilis bfmBAA NP_390285.1 16079461 Bacillus subtilis bfmBABNP_390284.1 16079460 Bacillus subtilis pdhD P21880.1 118672 Bacillussubtilis lpdV P09063.1 118677 Pseudomonas putida bkdB P09062.1 129044Pseudomonas putida bkdA1 NP_746515.1 26991090 Pseudomonas putida bkdA2NP_746516.1 26991091 Pseudomonas putida Bckdha NP_036914.1 77736548Rattus norvegicus Bckdhb NP_062140.1 158749538 Rattus norvegicus DbtNP_445764.1 158749632 Rattus norvegicus Dld NP_955417.1 40786469 Rattusnorvegicus

The pyruvate dehydrogenase complex, catalyzing the conversion ofpyruvate to acetyl-CoA, has also been extensively studied. In the E.coli enzyme, specific residues in the E1 component are responsible forsubstrate specificity (Bisswanger, H. J Biol. Chem. 256:815-822 (1981);Bremer, J. Eur. J Biochem. 8:535-540 (1969); Gong et al. J Biol. Chem.275:13645-13653 (2000)). As mentioned previously, enzyme engineeringefforts have improved the E. coli PDH enzyme activity under anaerobicconditions (Kim et al. Appl. Environ. Microbiol. 73:1766-1771 (2007);Kim J. Bacteriol. 190:3851-3858 (2008); Zhou et al. Biotechnol. Lett.30:335-342 (2008)). In contrast to the E. coli PDH, the B. subtiliscomplex is active and required for growth under anaerobic conditions(Nakano J. Bacteriol. 179:6749-6755 (1997)). The Klebsiella pneumoniaePDH, characterized during growth on glycerol, is also active underanaerobic conditions (Menzel et al. J. Biotechnol. 56:135-142 (1997)).Crystal structures of the enzyme complex from bovine kidney (Zhou et al.Proc. Natl. Acad. Sci. U.S.A. 98:14802-14807 (2001)) and the E2catalytic domain from Azotobacter vinelandii are available (Mattevi etal. Science 255:1544-1550 (1992)). Some mammalian PDH enzymes complexescan react on alternate substrates such as 2-oxobutanoate, althoughcomparative kinetics of Rattus norvegicus PDH and BCKAD indicate thatBCKAD has higher activity on 2-oxobutanoate as a substrate (Paxton etal. Biochem. J. 234:295-303 (1986)).

Gene Accession No. GI No. Organism aceE NP_414656.1 16128107 Escherichiacoli str. K12 substr. MG1655 aceF NP_414657.1 16128108 Escherichia colistr. K12 substr. MG1655 lpd NP_414658.1 16128109 Escherichia coli str.K12 substr. MG1655 pdhA P21881.1 3123238 Bacillus subtilis pdhB P21882.1129068 Bacillus subtilis pdhC P21883.2 129054 Bacillus subtilis pdhDP21880.1 118672 Bacillus subtilis aceE YP_001333808.1 152968699Klebsiella pneumonia MGH78578 aceF YP_001333809.1 152968700 Klebsiellapneumonia MGH78578 lpdA YP_001333810.1 152968701 Klebsiella pneumoniaMGH78578 Pdha1 NP_001004072.2 124430510 Rattus norvegicus Pdha2NP_446446.1 16758900 Rattus norvegicus Dlat NP_112287.1 78365255 Rattusnorvegicus Dld NP_955417.1 40786469 Rattus norvegicus

As an alternative to the large multienzyme 2-keto-acid dehydrogenasecomplexes described above, some anaerobic organisms utilize enzymes inthe 2-ketoacid oxidoreductase family (OFOR) to catalyze acylatingoxidative decarboxylation of 2-keto-acids. Unlike the dehydrogenasecomplexes, these enzymes contain iron-sulfur clusters, utilize differentcofactors, and use ferredoxin or flavodixin as electron acceptors inlieu of NAD(P)H. While most enzymes in this family are specific topyruvate as a substrate (POR) some 2-keto-acid:ferredoxinoxidoreductases have been shown to accept a broad range of 2-ketoacidsas substrates including alpha-ketoglutarate and 2-oxobutanoate (Fukudaand Wakagi Biochim. Biophys. Acta 1597:74-80 (2002); Zhang et al. J.Biochem. 120:587-599 (1996)). One such enzyme is the OFOR from thethermoacidophilic archaeon Sulfolobus tokodaii 7, which contains analpha and beta subunit encoded by gene ST2300 (Fukuda and WakagiBiochim. Biophys. Acta 1597:74-80 (2002); Zhang et al. J. Biochem.120:587-599 (1996)). A plasmid-based expression system has beendeveloped for efficiently expressing this protein in E. coli (Fukuda etal. Eur. J. Biochem. 268:5639-5646 (2001)) and residues involved insubstrate specificity were determined (Fukuda and Wakagi Biochim.Biophys. Acta 1597:74-80 (2002)). Two OFORs from Aeropyrum pernix str.K1 have also been recently cloned into E. coli, characterized, and foundto react with a broad range of 2-oxoacids (Nishizawa et al. FEBS Lett.579:2319-2322 (2005)). The gene sequences of these OFOR candidates areavailable, although they do not have GenBank identifiers assigned todate. There is bioinformatic evidence that similar enzymes are presentin all archaea, some anaerobic bacteria and amitochondrial eukarya(Fukuda and Wakagi Biochim. Biophys. Acta 1597:74-80 (2005)). This classof enzyme is also interesting from an energetic standpoint, as reducedferredoxin could be used to generate NADH by ferredoxin-NAD reductase(Petitdemange et al. Biochim. Biophys. Acta 421:334-337 (1976)). Also,since most of the enzymes are designed to operate under anaerobicconditions, less enzyme engineering may be required relative to enzymesin the 2-keto-acid dehydrogenase complex family for activity in ananaerobic environment.

Gene Accession No. GI No. Organism ST2300 NP_378302.1 15922633Sulfolobus tokodaii 71.2.1.d-Oxidoreductase (Phosphorylating/Dephosphorylating)

Exemplary enzymes in this class include glyceraldehyde 3-phosphatedehydrogenase which converts glyceraldehyde-3-phosphate into D-glycerate1,3-bisphosphate (for example, E. coli gapA (Branlant and Branlant Eur.J. Biochem. 150:61-66 (1985)), aspartate-semialdehyde dehydrogenasewhich converts L-aspartate-4-semialdehyde into L-4-aspartyl-phosphate(for example, E. coli asd (Biellmann et al. Eur. J. Biochem. 104:53-58(1980)), N-acetyl-gamma-glutamyl-phosphate reductase which convertsN-acetyl-L-glutamate-5-semialdehyde into N-acetyl-L-glutamyl-5-phosphate(for example, E. coli argC (Parsot et al. Gene 68:275-283 (1988)), andglutamate-5-semialdehyde dehydrogenase which convertsL-glutamate-5-semialdehyde into L-glutamyl-5-phospate (for example, E.coli proA (Smith et al. J. Bacteriol. 157:545-551 (1984)).

Gene Accession No. GI No. Organism gapA P0A9B2.2 71159358 Escherichiacoli asd NP_417891.1 16131307 Escherichia coli argC NP_418393.1 16131796Escherichia coli proA NP_414778.1 16128229 Escherichia coli1.3.1.a-Oxidoreductase Operating on CH—CH Donors

An exemplary enoyl-CoA reductase is the gene product of bcd from C.acetobutylicum (Atsumi et al. Metab Eng (2007); Boynton et al. Journalof Bacteriology 178:3015-3024 (1996), which naturally catalyzes thereduction of crotonyl-CoA to butyryl-CoA. Activity of this enzyme can beenhanced by expressing bcd in conjunction with expression of the C.acetobutylicum etfAB genes, which encode an electron transferflavoprotein. An additional candidate for the enoyl-CoA reductase stepis the mitochondrial enoyl-CoA reductase from E. gracilis (Hoffmeisteret al. Journal of Biological Chemistry 280:4329-4338 (2005)). Aconstruct derived from this sequence following the removal of itsmitochondrial targeting leader sequence was cloned in E. coli resultingin an active enzyme (Hoffmeister et al., supra, (2005)). This approachis well known to those skilled in the art of expressing eukarytoticgenes, particularly those with leader sequences that may target the geneproduct to a specific intracellular compartment, in prokaryoticorganisms. A close homolog of this gene, TDE0597, from the prokaryoteTreponema denticola represents a third enoyl-CoA reductase which hasbeen cloned and expressed in E. coli (Tucci and Martin FEBS Letters581:1561-1566 (2007)).

Gene Accession No. GI No. Organism bcd NP_349317.1 15895968 Clostridiumacetobutylicum etfA NP_349315.1 15895966 Clostridium acetobutylicum etfBNP_349316.1 15895967 Clostridium acetobutylicum TER Q5EU90.1 62287512Euglena gracilis TDE0597 NP_971211.1 42526113 Treponema denticola

Exemplary 2-enoate reductase (EC 1.3.1.31) enzymes are known to catalyzethe NADH-dependent reduction of a wide variety of α, β-unsaturatedcarboxylic acids and aldehydes (Rohdich et al. J. Biol. Chem.276:5779-5787 (2001)). 2-Enoate reductase is encoded by enr in severalspecies of Clostridia (Giesel and Simon Arch Microbiol. 135(1): p. 51-57(2001) including C. tyrobutyricum, and C. thermoaceticum (now calledMoorella thermoaceticum) (Rohdich et al., supra, (2001)). In therecently published genome sequence of C. kluyveri, 9 coding sequencesfor enoate reductases have been reported, out of which one has beencharacterized (Seedorf et al. Proc Natl Acad Sci U.S.A. 105(6):2128-33(2008)). The enr genes from both C. tyrobutyricum and C. thermoaceticumhave been cloned and sequenced and show 59% identity to each other. Theformer gene is also found to have approximately 75% similarity to thecharacterized gene in C. kluyveri (Giesel and Simon Arch Microbiol135(1):51-57 (1983)). It has been reported based on these sequenceresults that enr is very similar to the dienoyl CoA reductase in E. coli(fadH) (163 Rohdich et al., supra (2001)). The C. thermoaceticum enrgene has also been expressed in an enzymatically active form in E. coli(163 Rohdich et al., supra (2001)).

Gene Accession No. GI No. Organism fadH NP_417552.1 16130976 Escherichiacoli enr ACA54153.1 169405742 Clostridium botulinum A3 str enrCAA71086.1 2765041 Clostridium tyrobutyricum enr CAA76083.1 3402834Clostridium kluyveri enr YP_430895.1 83590886 Moorella thermoacetica1.4.1.a-Oxidoreductase Operating on Amino Acids

Most oxidoreductases operating on amino acids catalyze the oxidativedeamination of alpha-amino acids with NAD+ or NADP+ as acceptor.Exemplary oxidoreductases operating on amino acids include glutamatedehydrogenase (deaminating), encoded by gdhA, leucine dehydrogenase(deaminating), encoded by ldh, and aspartate dehydrogenase(deaminating), encoded by nadX. The gdhA gene product from Escherichiacoli (Korber et al. J. Mol. Biol. 234:1270-1273 (1993); McPherson andWootton Nucleic. Acids Res. 11:5257-5266 (1983)), gdh from Thermotogamaritima (Kort et al. Extremophiles 1:52-60 (1997); Lebbink, et al. J.Mol. Biol. 280:287-296 (1998)); Lebbink et al. J. Mol. Biol. 289:357-369(1999)), and gdhA1 from Halobacterium salinarum (Ingoldsby et al. Gene349:237-244 (2005)) catalyze the reversible interconversion of glutamateto 2-oxoglutarate and ammonia, while favoring NADP(H), NAD(H), or both,respectively. The ldh gene of Bacillus cereus encodes the LeuDH proteinthat has a wide of range of substrates including leucine, isoleucine,valine, and 2-aminobutanoate (Ansorge and Kula Biotechnol Bioeng.68:557-562 (2000); Stoyan et al. J. Biotechnol 54:77-80 (1997)). ThenadX gene from Thermotoga maritime encoding for the aspartatedehydrogenase is involved in the biosynthesis of NAD (Yang et al. J.Biol. Chem. 278:8804-8808 (2003)).

Gene Accession No. GI No. Organism gdhA P00370 118547 Escherichia coligdh P96110.4 6226595 Thermotoga maritima gdhA1 NP_279651.1 15789827Halobacterium salinarum ldh P0A393 61222614 Bacillus cereus nadXNP_229443.1 15644391 Thermotoga maritima

The lysine 6-dehydrogenase (deaminating), encoded by lysDH gene,catalyze the oxidative deamination of the ε-amino group of L-lysine toform 2-aminoadipate-6-semialdehyde, which in turn nonenzymaticallycyclizes to form Δ1-piperideine-6-carboxylate (Misono and Nagasaki J.Bacteriol. 150:398-401 (1982)). The lysDH gene from Geobacillusstearothermophilus encodes a thermophilic NAD-dependent lysine6-dehydrogenase (Heydari et al. Appl Environ. Microbiol 70:937-942(2004)). In addition, the lysDH gene from Aeropyrum pernix K1 isidentified through homology from genome projects.

Gene Accession No. GI No. Organism lysDH AB052732 13429872 Geobacillusstearothermophilus lysDH NP_147035.1 14602185 Aeropyrum pernix K1 ldhP0A393 61222614 Bacillus cereus2.3.1.a-Acyltransferase (Transferring Phosphate Group)

Exemplary phosphate transferring acyltransferases includephosphotransacetylase, encoded by pta, and phosphotransbutyrylase,encoded by ptb. The pta gene from E. coli encodes an enzyme that canconvert acetyl-CoA into acetyl-phosphate, and vice versa (Suzuki, T.Biochim. Biophys. Acta 191:559-569 (1969)). This enzyme can also utilizepropionyl-CoA instead of acetyl-CoA forming propionate in the process(Hesslinger et al. Mol. Microbiol 27:477-492 (1998)). Similarly, the ptbgene from C. acetobutylicum encodes an enzyme that can convertbutyryl-CoA into butyryl-phosphate (Walter et al. Gene 134(1): p. 107-11(1993)); Huang et al. J Mol Microbiol Biotechnol 2(1): p. 33-38 (2000).Additional ptb genes can be found in butyrate-producing bacterium L2-50(Louis et al. J. Bacteriol. 186:2099-2106 (2004)) and Bacillusmegaterium (Vazquez et al. Curr. Microbiol 42:345-349 (2001)).

Gene Accession No. GI No. Organism pta NP_416800.1 16130232 Escherichiacoli ptb NP_349676 15896327 Clostridium acetobutylicum ptb AAR19757.138425288 butyrate-producing bacterium L2-50 ptb CAC07932.1 10046659Bacillus megaterium2.6.1.a-Aminotransferase

Aspartate aminotransferase transfers an amino group from aspartate toalpha-ketoglutarate, forming glutamate and oxaloacetate. This conversionis catalyzed by, for example, the gene products of aspC from Escherichiacoli (Yagi et al. FEBS Lett. 100:81-84 (1979); Yagi et al. MethodsEnzymol. 113:83-89 (1985)), AAT2 from Saccharomyces cerevisiae (Yagi etal. J. Biochem. 92:35-43 (1982)) and ASPS from Arabidopsis thaliana (48,108, 225 48. de la et al. Plant J46:414-425 (2006); Kwok and Hanson JExp. Bot. 55:595-604 (2004); Wilkie and Warren Protein Expr. Purif.12:381-389 (1998)). Valine aminotransferase catalyzes the conversion ofvaline and pyruvate to 2-ketoisovalerate and alanine The E. coli gene,avtA, encodes one such enzyme (Whalen and Berg J. Bacteriol. 150:739-746(1982)). This gene product also catalyzes the amination ofα-ketobutyrate to generate α-aminobutyrate, although the amine donor inthis reaction has not been identified (Whalen and Berg J. Bacteriol.158:571-574 (1984)). The gene product of the E. coli serC catalyzes tworeactions, phosphoserine aminotransferase and phosphohydroxythreonineaminotransferase (Lam and Winkler J. Bacteriol. 172:6518-6528 (1990)),and activity on non-phosphorylated substrates could not be detected(Drewke et al. FEBS. Lett. 390:179-182 (1996)).

Gene Accession No. GI No. Organism aspC NP_415448.1 16128895 Escherichiacoli AAT2 P23542.3 1703040 Saccharomyces cerevisiae ASP5 P46248.220532373 Arabidopsis thaliana avtA YP_026231.1 49176374 Escherichia coliserC NP_415427.1 16128874 Escherichia coli

Cargill has developed a beta-alanine/alpha-ketoglutarateaminotransferase for producing 3-HP from beta-alanine viamalonyl-semialdehyde (PCT/US2007/076252 (Jessen et al)). The geneproduct of SkPYD4 in Saccharomyces kluyveri was also shown topreferentially use beta-alanine as the amino group donor (Andersen etal. FEBS. J. 274:1804-1817 (2007)). SkUGA1 encodes a homologue ofSaccharomyces cerevisiae GABA aminotransferase, UGA1 (Ramos et al. Eur.J. Biochem. 149:401-404 (1985)), whereas SkPYD4 encodes an enzymeinvolved in both β-alanine and GABA transamination (Andersen et al.FEBS. J. 274:1804-1817 (2007)). 3-Amino-2-methylpropionate transaminasecatalyzes the transformation from methylmalonate semialdehyde to3-amino-2-methylpropionate. The enzyme has been characterized in Rattusnorvegicus and Sus scrofa and is encoded by Abat (Kakimoto et al.Biochim. Biophys. Acta 156:374-380 (1968); Tamaki et al. MethodsEnzymol. 324:376-389 (2000)). Enzyme candidates in other organisms withhigh sequence homology to 3-amino-2-methylpropionate transaminaseinclude Gta-1 in C. elegans and gabT in Bacillus subtilus. Additionally,one of the native GABA aminotransferases in E. coli, encoded by genegabT, has been shown to have broad substrate specificity (Liu et al.Biochemistry 43:10896-10905 (2004); Schulz et al. Appl Environ Microbiol56:1-6 (1990)). The gene product of puuE catalyzes the other4-aminobutyrate transaminase in E. coli (Kurihara et al. J. Biol. Chem.280:4602-4608 (2005)).

Gene Accession No. GI No. Organism SkyPYD4 ABF58893.1 98626772Saccharomyces kluyveri SkUGA1 ABF58894.1 98626792 Saccharomyces kluyveriUGA1 NP_011533.1 6321456 Saccharomyces cerevisiae Abat P50554.3122065191 Rattus norvegicus Abat P80147.2 120968 Sus scrofa Gta-1Q21217.1 6016091 Caenorhabditis elegans gabT P94427.1 6016090 Bacillussubtilus gabT P22256.1 120779 Escherichia coli K12 puuE NP_415818.116129263 Escherichia coli K12

The X-ray crystal structures of E. coli 4-aminobutyrate transaminaseunbound and bound to the inhibitor were reported (Liu et al.Biochemistry 43:10896-10905 (2004)). The substrates binding andsubstrate specificities were studied and suggested. The roles of activesite residues were studied by site-directed mutagenesis and X-raycrystallography (Liu et al. Biochemistry 44:2982-2992 (2005)). Based onthe structural information, attempt was made to engineer E. coli4-aminobutyrate transaminase with novel enzymatic activity. Thesestudies provide a base for evolving transaminase activity for BDOpathways.

2.7.2.a-Phosphotransferase, Carboxyl Group Acceptor

Exemplary kinases include the E. coli acetate kinase, encoded by ackA(Skarstedt and Silverstein J. Biol. Chem. 251:6775-6783 (1976)), the C.acetobutylicum butyrate kinases, encoded by buk1 and buk2 (Walter et al.Gene 134(1):107-111 (1993) (Huang et al. J Mol Microbiol Biotechnol2(1):33-38 (2000)], and the E. coli gamma-glutamyl kinase, encoded byproB (Smith et al. J. Bacteriol. 157:545-551 (1984)). These enzymesphosphorylate acetate, butyrate, and glutamate, respectively. The ackAgene product from E. coli also phosphorylates propionate (Hesslinger etal. Mol. Microbiol 27:477-492 (1998)).

Gene Accession No. GI No. Organism ackA NP_416799.1 16130231 Escherichiacoli buk1 NP_349675 15896326 Clostridium acetobutylicum buk2 Q97II120137415 Clostridium acetobutylicum proB NP_414777.1 16128228Escherichia coli2.8.3.a-Coenzyme-A Transferase

In the CoA-transferase family, E. coli enzyme acyl-CoA:acetate-CoAtransferase, also known as acetate-CoA transferase (EC 2.8.3.8), hasbeen shown to transfer the CoA moiety to acetate from a variety ofbranched and linear acyl-CoA substrates, including isobutyrate (Matthiesand Schink Appl Environ Microbiol 58:1435-1439 (1992)), valerate(Vanderwinkel et al. Biochem. Biophys. Res Commun. 33:902-908 (1968))and butanoate (Vanderwinkel, supra (1968)). This enzyme is encoded byatoA (alpha subunit) and atoD (beta subunit) in E. coli sp. K12 (Korolevet al. Acta Crystallogr. D Biol Crystallogr. 58:2116-2121 (2002);Vanderwinkel, supra (1968)) and actA and cg0592 in Corynebacteriumglutamicum ATCC 13032 (Duncan et al. Appl Environ Microbiol 68:5186-5190(2002)). Additional genes found by sequence homology include atoD andatoA in Escherichia coli UT189.

Gene Accession No. GI No. Organism atoA P76459.1 2492994 Escherichiacoli K12 atoD P76458.1 2492990 Escherichia coli K12 actA YP_226809.162391407 Corynebacterium glutamicum ATCC 13032 cg0592 YP_224801.162389399 Corynebacterium glutamicum ATCC 13032 atoA ABE07971.1 91073090Escherichia coli UT189 atoD ABE07970.1 91073089 Escherichia coli UT189

Similar transformations are catalyzed by the gene products of cat1,cat2, and cat3 of Clostridium kluyveri which have been shown to exhibitsuccinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA acetyltransferaseactivity, respectively (Seedorf et al. Proc Natl Acad Sci U.S.A.105(6):2128-2133 (2008); Sohling and Gottschalk J Bacteriol178(3):871-880 (1996)].

Gene Accession No. GI No. Organism cat1 P38946.1 729048 Clostridiumkluyveri cat2 P38942.2 1705614 Clostridium kluyveri cat3 EDK35586.1146349050 Clostridium kluyveri

The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from anaerobicbacterium Acidaminococcus fermentans reacts with diacid glutaconyl-CoAand 3-butenoyl-CoA (Mack and Buckel FEBS Lett. 405:209-212 (1997)). Thegenes encoding this enzyme are gctA and gctB. This enzyme has reducedbut detectable activity with other CoA derivatives includingglutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckelet al. Eur. J. Biochem. 118:315-321 (1981)). The enzyme has been clonedand expressed in E. coli (Mac et al. Eur. J. Biochem. 226:41-51 (1994)).

Gene Accession No. GI No. Organism gctA CAA57199.1 559392Acidaminococcus fermentans gctB CAA57200.1 559393 Acidaminococcusfermentans3.1.2.a-Thiolester Hydrolase (CoA Specific)

In the CoA hydrolase family, the enzyme 3-hydroxyisobutyryl-CoAhydrolase is specific for 3-HIBCoA and has been described to efficientlycatalyze the desired transformation during valine degradation (Shimomuraet al. J Biol Chem 269:14248-14253 (1994)). Genes encoding this enzymeinclude hibch of Rattus norvegicus (Shimomura et al., supra (1994);Shimomura et al. Methods Enzymol. 324:229-240 (2000) and Homo sapiens(Shimomura et al., supra, 2000). Candidate genes by sequence homologyinclude hibch of Saccharomyces cerevisiae and BC_2292 of Bacilluscereus.

Gene Accession No. GI No. Organism hibch Q5XIE6.2 146324906 Rattusnorvegicus hibch Q6NVY1.2 146324905 Homo sapiens hibch P28817.2 2506374Saccharomyces cerevisiae BC_2292 Q81DR3 81434808 Bacillus cereus

The conversion of adipyl-CoA to adipate can be carried out by anacyl-CoA hydrolase or equivalently a thioesterase. The top E. coli genecandidate is tesB (Naggert et al. J Biol. Chem. 266(17):11044-11050(1991)] which shows high similarity to the human acot8 which is adicarboxylic acid acetyltransferase with activity on adipyl-CoA (Westinet al. J Biol Chem 280(46): 38125-38132 (2005). This activity has alsobeen characterized in the rat liver (Deana, Biochem Int. 26(4): p.767-773 (1992)).

Gene Accession No. GI No. Organism tesB NP_414986 16128437 Escherichiacoli acot8 CAA15502 3191970 Homo sapiens acot8 NP_570112 51036669 Rattusnorvegicus

Other potential E. coli thiolester hydrolases include the gene productsof tesA (Bonner and Bloch, J Biol. Chem. 247(10):3123-3133 (1972)), ybgC(Kuznetsova et al., FEMS Microbiol Rev. 29(2):263-279 (2005); Zhuang etal., FEBS Lett. 516(1-3):161-163 (2002)) paaI (Song et al., J Biol.Chem. 281(16):11028-11038 (2006)), and ybdB (Leduc et al., J Bacteriol.189(19):7112-7126 (2007)).

Gene Accession No. GI No. Organism tesA NP_415027 16128478 Escherichiacoli ybgC NP_415264 16128711 Escherichia coli paaI NP_415914 16129357Escherichia coli ybdB NP_415129 16128580 Escherichia coli

Several eukaryotic acetyl-CoA hydrolases (EC 3.1.2.1) have broadsubstrate specificity. The enzyme from Rattus norvegicus brain (Robinsonet al. Biochem. Biophys. Res. Commun. 71:959-965 (1976)) can react withbutyryl-CoA, hexanoyl-CoA and malonyl-CoA.

Gene Accession No. GI No. Organism acot12 NP_570103.1 18543355 Rattusnorvegicus4.1.1.a-Carboxy-Lyase

An exemplary carboxy-lyase is acetolactate decarboxylase whichparticipates in citrate catabolism and branched-chain amino acidbiosynthesis, converting 2-acetolactate to acetoin. In Lactococcuslactis the enzyme is composed of six subunits, encoded by gene aldB, andis activated by valine, leucine and isoleucine (Goupil et al. Appl.Environ. Microbiol. 62:2636-2640 (1996); Goupil-Feuillerat et al. J.Bacteriol. 182:5399-5408 (2000)). This enzyme has been overexpressed andcharacterized in E. coli (Phalip et al. FEBS Lett. 351:95-99 (1994)). Inother organisms the enzyme is a dimer, encoded by aldC in Streptococcusthermophilus (Monnet et al. Lett. Appl. Microbiol. 36:399-405 (2003)),aldB in Bacillus brevis (Diderichsen et al. J. Bacteriol. 172:4315-4321(1990); Najmudin et al. Acta Crystallogr. D. Biol. Crystallogr.59:1073-1075 (2003)) and budA from Enterobacter aerogenes (Diderichsenet al. J. Bacteriol. 172:4315-4321 (1990)). The enzyme from Bacillusbrevis was cloned and overexpressed in Bacillus subtilis andcharacterized crystallographically (Najmudin et al. Acta Crystallogr. D.Biol. Crystallogr. 59:1073-1075 (2003)). Additionally, the enzyme fromLeuconostoc lactis has been purified and characterized but the gene hasnot been isolated (O'Sullivan et al. FEMS Microbiol. Lett. 194:245-249(2001)).

Gene Accession No. GI No. Organism aldB NP_267384.1 15673210 Lactococcuslactis aldC Q8L208 75401480 Streptococcus thermophilus aldB P23616.1113592 Bacillus brevis budA P05361.1 113593 Enterobacter aerogenes

Aconitate decarboxylase catalyzes the final step in itaconatebiosynthesis in a strain of Candida and also in the filamentous fungusAspergillus terreus (Bonnarme et al. J. Bacteriol. 177:3573-3578 (1995);Willke and Vorlop Appl Microbiol Biotechnol 56:289-295 (2001)). Althoughitaconate is a compound of biotechnological interest, the aconitatedecarboxylase gene or protein sequence has not been reported to date.

4-oxalocronate decarboxylase has been isolated from numerous organismsand characterized. Genes encoding this enzyme include dmpH and dmpE inPseudomonas sp. (strain 600) (Shingler et al. J. Bacteriol. 174:711-724(1992)), xylII and xylIII from Pseudomonas putida (Kato and Asano Arch.Microbiol 168:457-463 (1997); Lian and Whitman J. Am. Chem. Soc.116:10403-10411 (1994); Stanley et al. Biochemistry 39:3514 (2000)) andReut_B5691 and Reut_B5692 from Ralstonia eutropha JMP134 (Hughes et al.J. Bacteriol. 158:79-83 (1984)). The genes encoding the enzyme fromPseudomonas sp. (strain 600) have been cloned and expressed in E. coli(Shingler et al. J. Bacteriol. 174:711-724 (1992)).

Gene Accession No. GI No. Organism dmpH CAA43228.1 45685 Pseudomonas sp.CF600 dmpE CAA43225.1 45682 Pseudomonas sp. CF600 xylII YP_709328.1111116444 Pseudomonas putida xylIII YP_709353.1 111116469 Pseudomonasputida Reut_B5691 YP_299880.1 73539513 Ralstonia eutropha JMP134Reut_B5692 YP_299881.1 73539514 Ralstonia eutropha JMP134

An additional class of decarboxylases has been characterized thatcatalyze the conversion of cinnamate (phenylacrylate) and substitutedcinnamate derivatives to the corresponding styrene derivatives. Theseenzymes are common in a variety of organisms and specific genes encodingthese enzymes that have been cloned and expressed in E. coli are: pad 1from Saccharomyces cerevisae (Clausen et al. Gene 142:107-112 (1994)),pdc from Lactobacillus plantarum (Barthelmebs et al. Appl EnvironMicrobiol 67:1063-1069 (2001); Qi et al. Metab Eng 9:268-276 (2007);Rodriguez et al. J. Agric. Food Chem. 56:3068-3072 (2008)), pofK (pad)from Klebsiella oxytoca (Hashidoko et al. Biosci. Biotech. Biochem.58:217-218 (1994); Uchiyama et al. Biosci. Biotechnol. Biochem.72:116-123 (2008)), Pedicoccus pentosaceus (Barthelmebs et al. ApplEnviron Microbiol 67:1063-1069 (2001)), and padC from Bacillus subtilisand Bacillus pumilus (Lingen et al. Protein Eng 15:585-593 (2002)). Aferulic acid decarboxylase from Pseudomonas fluorescens also has beenpurified and characterized (Huang et al. J. Bacteriol. 176:5912-5918(1994)). Importantly, this class of enzymes have been shown to be stableand do not require either exogenous or internally bound co-factors, thusmaking these enzymes ideally suitable for biotransformations(Sariaslani, Annu. Rev. Microbiol. 61:51-69 (2007)).

Gene Accession No. GI No. Organism pad1 AB368798 188496948 SaccharomycesBAG32372.1 188496949 cerevisae pdc U63827 1762615, 1762616 LactobacillusAAC45282.1 plantarum pofK (pad) AB330293, 149941607, KlebsiellaBAF65031.1 149941608 oxytoca padC AF017117 2394281, 2394282 BacillusAAC46254.1 subtilis pad AJ276891 11322456, 11322458 PedicoccusCAC16794.1 pentosaceus pad AJ278683 11691809, 11691810 BacillusCAC18719.1 pumilus

Additional decarboxylase enzymes can form succinic semialdehyde fromalpha-ketoglutarate. These include the alpha-ketoglutarate decarboxylaseenzymes from Euglena gracilis (Shigeoka et al. Biochem. J. 282(Pt2):319-323 (1992); Shigeoka and Nakano Arch. Biochem. Biophys. 288:22-28(1991); Shigeoka and Nakano Biochem. J. 292 (Pt 2):463-467 (1993)),whose corresponding gene sequence has yet to be determined, and fromMycobacterium tuberculosis (Tian et al. Proc Natl Acad Sci U.S.A.102:10670-10675 (2005)). In addition, glutamate decarboxylase enzymescan convert glutamate into 4-aminobutyrate such as the products of theE. coli gadA and gadB genes (De Biase et al. Protein. Expr. Purif.8:430-438 (1993)).

Gene Accession No. GI No. Organism kgd O50463.4 160395583 Mycobacteriumtuberculosis gadA NP_417974 16131389 Escherichia coli gadB NP_41601016129452 Escherichia coliKeto-Acid Decarboxylases

Pyruvate decarboxylase (PDC, EC 4.1.1.1), also termed keto-aciddecarboxylase, is a key enzyme in alcoholic fermentation, catalyzing thedecarboxylation of pyruvate to acetaldehyde. This enzyme has a broadsubstrate range for aliphatic 2-keto acids including 2-ketobutyrate,2-ketovalerate, 3-hydroxypyruvate and 2-phenylpyruvate (Berg et al.Science 318:1782-1786 (2007)). The PDC from Zymomonas mobilus, encodedby pdc, has been a subject of directed engineering studies that alteredthe affinity for different substrates (Siegert et al. Protein Eng DesSel 18:345-357 (2005)). The PDC from Saccharomyces cerevisiae has alsobeen extensively studied, engineered for altered activity, andfunctionally expressed in E. coli (Killenberg-Jabs et al. Eur. J.Biochem. 268:1698-1704 (2001); Li and Jordan Biochemistry 38:10004-10012(1999); ter Schure et al. Appl. Environ. Microbiol. 64:1303-1307(1998)). The crystal structure of this enzyme is available(Killenberg-Jabs Eur. J. Biochem. 268:1698-1704 (2001)). Otherwell-characterized PDC candidates include the enzymes from Acetobacterpasteurians (Chandra et al. Arch. Microbiol. 176:443-451 (2001)) andKluyveromyces lactis (Krieger et al. Eur. J. Biochem. 269:3256-3263(2002)).

Gene Accession No. GI No. Organism pdc P06672.1 118391 Zymomonas mobiluspdc1 P06169 30923172 Saccharomyces cerevisiae pdc Q8L388 75401616Acetobacter pasteurians pdc1 Q12629 52788279 Kluyveromyces lactis

Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broadsubstrate range and has been the target of enzyme engineering studies.The enzyme from Pseudomonas putida has been extensively studied andcrystal structures of this enzyme are available (Hasson et al.Biochemistry 37:9918-9930 (1998); Polovnikova et al. Biochemistry42:1820-1830 (2003)). Site-directed mutagenesis of two residues in theactive site of the Pseudomonas putida enzyme altered the affinity (Km)of naturally and non-naturally occurring substrates (Siegert Protein EngDes Sel 18:345-357 (2005)). The properties of this enzyme have beenfurther modified by directed engineering (Lingen et al. Protein Eng15:585-593 (2002)); Lingen Chembiochem 4:721-726 (2003)). The enzymefrom Pseudomonas aeruginosa, encoded by mdlC, has also beencharacterized experimentally (Barrowman et al. FEMS Microbiology Letters34:57-60 (1986)). Additional gene candidates from Pseudomonas stutzeri,Pseudomonas fluorescens and other organisms can be inferred by sequencehomology or identified using a growth selection system developed inPseudomonas putida (Henning et al. Appl. Environ. Microbiol.72:7510-7517 (2006)).

Gene Accession No. GI No. Organism mdlC P20906.2 3915757 Pseudomonasputida mdlC Q9HUR2.1 81539678 Pseudomonas aeruginosa dpgB ABN80423.1126202187 Pseudomonas stutzeri ilvB-1 YP_260581.1 70730840 Pseudomonasfluorescens4.2.1.a-Hydro-Lyase

The 2-(hydroxymethyl)glutarate dehydratase of Eubacterium barkeri is anexemplary hydro-lyase. This enzyme has been studied in the context ofnicotinate catabolism and is encoded by hmd (Alhapel et al. Proc NatlAcad Sci USA 103:12341-12346 (2006)). Similar enzymes with high sequencehomology are found in Bacteroides capillosus, Anaerotruncus colihominis,and Natranaerobius thermophilius.

Gene Accession No. GI No. Organism hmd ABC88407.1 86278275 Eubacteriumbarkeri BACCAP_02294 ZP_02036683.1 154498305 Bacteroides capillosus ATCC29799 ANACOL_02527 ZP_02443222.1 167771169 Anaerotruncus colihominis DSM17241 NtherDRAFT_2368 ZP_02852366.1 169192667 Natranaerobiusthermophilus JW/NM-WN-LF

A second exemplary hydro-lyase is fumarate hydratase, an enzymecatalyzing the dehydration of malate to fumarate. A wealth of structuralinformation is available for this enzyme and researchers havesuccessfully engineered the enzyme to alter activity, inhibition andlocalization (Weaver, T. Acta Crystallogr. D Biol Crystallogr.61:1395-1401 (2005)). Additional fumarate hydratases include thoseencoded by fumC from Escherichia coli (Estevez et al. Protein Sci.11:1552-1557 (2002); Hong and Lee Biotechnol. Bioprocess Eng. 9:252-255(2004); Rose and Weaver Proc Natl Acad Sci U S.A 101:3393-3397 (2004)),Campylobacter jejuni (Smith et al. Int. J. Biochem. Cell Biol 31:961-975(1999)) and Thermus thermophilus (Mizobata et al. Arch. Biochem.Biophys. 355:49-55 (1998)), and fumH from Rattus norvegicus (Kobayashiet al. J. Biochem. 89:1923-1931 (1981)). Similar enzymes with highsequence homology include fuml from Arabidopsis thaliana and fumC fromCorynebacterium glutamicum.

Gene Accession No. GI No. Organism fumC P05042.1 120601 Escherichia coliK12 fumC O69294.1 9789756 Campylobacter jejuni fumC P84127 75427690Thermus thermophilus fumH P14408.1 120605 Rattus norvegicus fum1P93033.2 39931311 Arabidopsis thaliana fumC Q8NRN8.1 39931596Corynebacterium glutamicum

Citramalate hydrolyase, also called 2-methylmalate dehydratase, converts2-methylmalate to mesaconate. 2-Methylmalate dehydratase activity wasdetected in Clostridium tetanomorphum, Morganella morganii, Citrobacteramalonaticus in the context of the glutamate degradation VI pathway(Kato and Asano Arch. Microbiol 168:457-463 (1997)); however the genesencoding this enzyme have not been sequenced to date.

The gene product of crt from C. acetobutylicum catalyzes the dehydrationof 3-hydroxybutyryl-CoA to crotonyl-CoA (Atsumi et al. Metab Eng.; 29(2007)); Boynton et al. Journal of Bacteriology 178:3015-3024 (1996)).The enoyl-CoA hydratases, phaA and phaB, of P. putida are believed tocarry out the hydroxylation of double bonds during phenylacetatecatabolism; (Olivera et al. Proc Natl Acad Sci USA 95(11):6419-6424(1998)). The paaA and paaB from P. fluorescens catalyze analogoustransformations (14 Olivera et al., supra, 1998). Lastly, a number ofEscherichia coli genes have been shown to demonstrate enoyl-CoAhydratase functionality including maoC (Park and Lee J Bacteriol185(18):5391-5397 (2003)), paaF (Park and Lee Biotechnol Bioeng.86(6):681-686 (2004a)); Park and Lee Appl Biochem Biotechnol. 113-116:335-346 (2004b)); Ismail et al. Eur J Biochem 270(14):p. 3047-3054(2003), and paaG (Park and Lee, supra, 2004; Park and Lee supra, 2004b;Ismail et al., supra, 2003).

Gene Accession No. GI No. Organism maoC NP_415905.1 16129348 Escherichiacoli paaF NP_415911.1 16129354 Escherichia coli paaG NP_415912.116129355 Escherichia coli crt NP_349318.1 15895969 Clostridiumacetobutylicum paaA NP_745427.1 26990002 Pseudomonas putida paaBNP_745426.1 26990001 Pseudomonas putida phaA ABF82233.1 106636093Pseudomonas fluorescens phaB ABF82234.1 106636094 Pseudomonasfluorescens

The E. coli genes fadA and fadB encode a multienzyme complex thatexhibits ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, andenoyl-CoA hydratase activities (Yang et al. Biochemistry 30(27): p.6788-6795 (1991); Yang et al. J Biol Chem 265(18): p. 10424-10429(1990); Yang et al. J Biol Chem 266(24): p. 16255 (1991); Nakahigashiand Inokuchi Nucleic Acids Res 18(16): p. 4937 (1990)). The fadI andfadJ genes encode similar functions and are naturally expressed onlyanaerobically (Campbell et al. Mol Microbiol 47(3): p. 793-805 (2003). Amethod for producing poly[(R)-3-hydroxybutyrate] in E. coli thatinvolves activating fadB (by knocking out a negative regulator, fadR)and co-expressing a non-native ketothiolase (phaA from Ralstoniaeutropha) has been described previously (Sato et al. J Biosci Bioeng103(1): 38-44 (2007)). This work clearly demonstrates that a β-oxidationenzyme, in particular the gene product of fadB which encodes both3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase activities, canfunction as part of a pathway to produce longer chain molecules fromacetyl-CoA precursors.

Gene Accession No. GI No. Organism fadA YP_026272.1 49176430 Escherichiacoli fadB NP_418288.1 16131692 Escherichia coli fadI NP_416844.116130275 Escherichia coli fadJ NP_416843.1 16130274 Escherichia colifadR NP_415705.1 16129150 Escherichia coli4.3.1.a-Ammonia-Lyase

Aspartase (EC 4.3.1.1), catalyzing the deamination of aspartate tofumarate, is a widespread enzyme in microorganisms, and has beencharacterized extensively (Viola, R. E. Adv. Enzymol. Relat Areas Mol.Biol. 74:295-341 (2000)). The crystal structure of the E. coliaspartase, encoded by aspA, has been solved (Shi et al. Biochemistry36:9136-9144 (1997)). The E. coli enzyme has also been shown to reactwith alternate substrates aspartatephenylmethylester, asparagine,benzyl-aspartate and malate (Ma et al. Ann N.Y. Acad Sci 672:60-65(1992)). In a separate study, directed evolution was been employed onthis enzyme to alter substrate specificity (Asano et al. Biomol. Eng22:95-101 (2005)). Enzymes with aspartase functionality have also beencharacterized in Haemophilus influenzae (Sjostrom et al. Biochim.Biophys. Acta 1324:182-190 (1997)), Pseudomonas fluorescens (Takagi etal. J. Biochem. 96:545-552 (1984)), Bacillus subtilus (Sjostrom et al.Biochim. Biophys. Acta 1324:182-190 (1997)) and Serratia marcescens(Takagi and Kisumi J Bacteriol. 161:1-6 (1985)).

Gene Accession No. GI No. Organism aspA NP_418562 90111690 Escherichiacoli K12 subsp. MG1655 aspA P44324.1 1168534 Haemophilus influenzae aspAP07346.1 114273 Pseudomonas fluorescens ansB P26899.1 114271 Bacillussubtilus aspA P33109.1 416661 Serratia marcescens

3-methylaspartase (EC 4.3.1.2), also known as beta-methylaspartase or3-methylaspartate ammonia-lyase, catalyzes the deamination ofthreo-3-methylasparatate to mesaconate. The 3-methylaspartase fromClostridium tetanomorphum has been cloned, functionally expressed in E.coli, and crystallized (Asuncion et al. Acta Crystallogr. D BiolCrystallogr. 57:731-733 (2001); Asuncion et al. J Biol. Chem.277:8306-8311 (2002); Botting et al. Biochemistry 27:2953-2955 (1988);Goda et al. Biochemistry 31:10747-10756 (1992). In Citrobacteramalonaticus, this enzyme is encoded by BAA28709 (Kato and Asano Arch.Microbiol 168:457-463 (1997)). 3-Methylaspartase has also beencrystallized from E. coli YG1002 (Asano and Kato FEMS Microbiol Lett.118:255-258 (1994)) although the protein sequence is not listed inpublic databases such as GenBank. Sequence homology can be used toidentify additional candidate genes, including CTC_02563 in C. tetaniand ECs0761 in Escherichia coli O157:H7.

Gene Accession No. GI No. Organism MAL AAB24070.1 259429 Clostridiumtetanomorphum BAA28709 BAA28709.1 3184397 Citrobacter amalonaticusCTC_02563 NP_783085.1 28212141 Clostridium tetani ECs0761 BAB34184.113360220 Escherichia coli O157:H7 str. Sakai

Ammonia-lyase enzyme candidates that form enoyl-CoA products includebeta-alanyl-CoA ammonia-lyase (EC 4.3.1.6), which deaminatesbeta-alanyl-CoA, and 3-aminobutyryl-CoA ammonia-lyase (EC 4.3.1.14). Twobeta-alanyl-CoA ammonia lyases have been identified and characterized inClostridium propionicum (Herrmann et al. FEBS J. 272:813-821 (2005)). Noother beta-alanyl-CoA ammonia lyases have been studied to date, but genecandidates can be identified by sequence similarity. One such candidateis MXAN_4385 in Myxococcus xanthus.

Gene Accession No. GI No. Organism ac12 CAG29275.1 47496504 Clostridiumpropionicum acl1 CAG29274.1 47496502 Clostridium propionicum MXAN_4385YP_632558.1 108756898 Myxococcus xanthus5.3.3.a-Isomerase

The 4-hydroxybutyryl-CoA dehydratases from both Clostridiumaminobutyrium and C. kluyveri catalyze the reversible conversion of4-hydroxybutyryl-CoA to crotonyl-CoA and posses an intrinsicvinylacetyl-CoA Δ-isomerase activity (Scherf and Buckel Eur. J Biochem.215:421-429 (1993); Scherf et al. Arch. Microbiol 161:239-245 (1994)).Both native enzymes were purified and characterized, including theN-terminal amino acid sequences (Scherf and Buckel, supra, 1993; Scherfet al., supra, 1994). The abfD genes from C. aminobutyrium and C.kluyveri match exactly with these N-terminal amino acid sequences, thusare encoding the 4-hydroxybutyryl-CoA dehydratases/vinylacetyl-CoAΔ-isomerase. In addition, the abfD gene from Porphyromonas gingivalisATCC 33277 is identified through homology from genome projects.

Gene Accession No. GI No. Organism abfD YP_001396399.1 153955634Clostridium kluyveri DSM 555 abfD P55792 84028213 Clostridiumaminobutyricum abfD YP_001928843 188994591 Porphyromonas gingivalis ATCC332775.4.3.a-Aminomutase

Lysine 2,3-aminomutase (EC 5.4.3.2) is an exemplary aminomutase thatconverts lysine to (3S)-3,6-diaminohexanoate, shifting an amine groupfrom the 2- to the 3-position. The enzyme is found in bacteria thatferment lysine to acetate and butyrate, including as Fusobacteriumnuleatum (kamA) (Barker et al. J. Bacteriol. 152:201-207 (1982)) andClostridium subterminale (kamA) (Chirpich et al. J. Biol. Chem.245:1778-1789 (1970)). The enzyme from Clostridium subterminale has beencrystallized (Lepore et al. Proc. Natl. Acad. Sci. U.S.A 102:13819-13824(2005)). An enzyme encoding this function is also encoded by yodO inBacillus subtilus (Chen et al. Biochem. J. 348 Pt 3:539-549 (2000)). Theenzyme utilizes pyridoxal 5′-phosphate as a cofactor, requiresactivation by S-Adenosylmethoionine, and is stereoselective, reactingwith the only with L-lysine. The enzyme has not been shown to react withalternate substrates.

Gene Accession No. GI No. Organism yodO O34676.1 4033499 Bacillussubtilus kamA Q9XBQ8.1 75423266 Clostridium subterminale kamA Q8RHX481485301 Fusobacterium nuleatum subsp. nuleatum

A second aminomutase, beta-lysine 5,6-aminomutase (EC 5.4.3.3),catalyzes the next step of lysine fermentation to acetate and butyrate,which transforms (3S)-3,6-diaminohexanoate to(3S,5S)-3,5-diaminohexanoate, shifting a terminal amine group from the6- to the 5-position. This enzyme also catalyzes the conversion oflysine to 2,5-diaminohexanoate and is also called lysine-5,6-aminomutase(EC 5.4.3.4). The enzyme has been crystallized in Clostridiumsticklandii (kamD, kamE) (Berkovitch et al. Proc. Natl. Acad. Sci. U.S.A101:15870-15875 (2004)). The enzyme from Porphyromonas gingivalis hasalso been characterized (Tang et al. Biochemistry 41:8767-8776 (2002)).

Gene Accession No. GI No. Organism kamD AAC79717.1  3928904 Clostridiumsticklandii kamE AAC79718.1  3928905 Clostridium sticklandii kamDNC_002950.2 34539880, Porphyromonas gingivalis 34540809 W83 kamENC_002950.2 34539880, Porphyromonas gingivalis 34540810 W83

Ornithine 4,5-aminomutase (EC 5.4.3.5) converts D-ornithine to2,4-diaminopentanoate, also shifting a terminal amine to the adjacentcarbon. The enzyme from Clostridium sticklandii is encoded by two genes,oraE and oras, and has been cloned, sequenced and expressed in E. coli(Chen et al. J. Biol. Chem. 276:44744-44750 (2001)). This enzyme has notbeen characterized in other organisms to date.

Gene Accession No. GI No. Organism oraE AAK72502 17223685 Clostridiumsticklandii oraS AAK72501 17223684 Clostridium sticklandii

Tyrosine 2,3-aminomutase (EC 5.4.3.6) participates in tyrosinebiosynthesis, reversibly converting tyrosine to3-amino-3-(4-hdyroxyphenyl)propanoate by shifting an amine from the 2-to the 3-position. In Streptomyces globisporus the enzyme has also beenshown to react with tyrosine derivatives (Christenson et al.Biochemistry 42:12708-12718 (2003)). Sequence information is notavailable.

Leucine 2,3-aminomutase (EC 5.4.3.7) converts L-leucine to beta-leucineduring leucine degradation and biosynthesis. An assay for leucine2,3-aminomutase detected activity in many organisms (Poston, J. M.Methods Enzymol. 166:130-135 (1988)) but genes encoding the enzyme havenot been identified to date.

Cargill has developed a novel 2,3-aminomutase enzyme to convertL-alanine to β-alanine, thus creating a pathway from pyruvate to 3-HP infour biochemical steps (Liao et al., U.S. Publication No. 2005-0221466).

6.2.1.a- Acid-Thiol Ligase

An exemplary acid-thiol ligase is the gene products of sucCD of E. coliwhich together catalyze the formation of succinyl-CoA from succinatewith the concaminant consumption of one ATP, a reaction which isreversible in vivo (Buck et al. Biochemistry 24(22): p. 6245-6252(1985)). Additional exemplary CoA-ligases include the ratdicarboxylate-CoA ligase for which the sequence is yet uncharacterized(Vamecq et al. Biochem J. 230(3): p. 683-693 (1985)), either of the twocharacterized phenylacetate-CoA ligases from P. chrysogenum(Lamas-Maceiras et al. Biochem J 395(1):147-155 (2006); Wang et al.Biochem Biophys Res Commun, 360(2):453-458 (2007)), thephenylacetate-CoA ligase from Pseudomonas putida (Martinez-Blanco et al.J Biol. Chem. 265(12):7084-7090 (1990)), and the 6-carboxyhexanoate-CoAligase from Bacillus subtilis (Bower et al. J Bacteriol178(14):4122-4130 (1996)).

Gene Accession No. GI No. Organism sucC NP_415256.1 16128703 Escherichiacoli sucD AAC73823.1 1786949 Escherichia coli phl CAJ15517.1 77019264Penicillium chrysogenum phlB ABS19624.1 152002983 Penicilliumchrysogenum paaF AAC24333.2 22711873 Pseudomonas putida bioW NP_390902.250812281 Bacillus subtilis

EXAMPLE V Exemplary BDO Pathway from Succinyl-CoA

This example describes exemplary BDO pathways from succinyl-CoA.

BDO pathways from succinyl-CoA are described herein and have beendescribed previously (see U.S. application Ser. No. 12/049,256, filedMar. 14, 2008, and PCT application Ser. No. U.S.08/57168, filed Mar. 14,2008, each of which is incorporated herein by reference). Additionalpathways are shown in FIG. 8A. Enzymes of such exemplary BDO pathwaysare listed in Table 15, along with exemplary genes encoding theseenzymes.

Briefly, succinyl-CoA can be converted to succinic semialdehyde bysuccinyl-CoA reductase (or succinate semialdehyde dehydrogenase) (EC1.2.1.b). Succinate semialdehyde can be converted to 4-hydroxybutyrateby 4-hydroxybutyrate dehydrogenase (EC 1.1.1.a), as previouslydescribed. Alternatively, succinyl-CoA can be converted to4-hydroxybutyrate by succinyl-CoA reductase (alcohol forming) (EC1.1.1.c). 4-Hydroxybutyrate can be converted to 4-hydroxybutyryl-CoA by4-hydroxybutyryl-CoA transferase (EC 2.8.3.a), as previously described,or by 4-hydroxybutyryl-CoA hydrolase (EC 3.1.2.a) or4-hydroxybutyryl-CoA ligase (or 4-hydroxybutyryl-CoA synthetase) (EC6.2.1.a). Alternatively, 4-hydroxybutyrate can be converted to4-hydroxybutyryl-phosphate by 4-hydroxybutyrate kinase (EC 2.7.2.a), aspreviously described. 4-Hydroxybutyryl-phosphate can be converted to4-hydroxybutyryl-CoA by phosphotrans-4-hydroxybutyrylase (EC 2.3.1.a),as previously described. Alternatively, 4-hydroxybutyryl-phosphate canbe converted to 4-hydroxybutanal by 4-hydroxybutanal dehydrogenase(phosphorylating) (EC 1.2.1.d). 4-Hydroxybutyryl-CoA can be converted to4-hydroxybutanal by 4-hydroxybutyryl-CoA reductase (or 4-hydroxybutanaldehydrogenase) (EC 1.2.1.b). Alternatively, 4-hydroxybutyryl-CoA can beconverted to 1,4-butanediol by 4-hydroxybutyryl-CoA reductase (alcoholforming) (EC 1.1.1.c). 4-Hydroxybutanal can be converted to1,4-butanediol by 1,4-butanediol dehydrogenase (EC 1.1.1.a), aspreviously described.

TABLE 15 BDO pathway from succinyl-CoA. EC Desired Desired Gene GenBankID Known FIG. class substrate product Enzyme name name (if available)Organism Substrates 8A 1.2.1.b succinyl-CoA succinic succinyl-CoA sucDP38947.1 Clostridium succinyl-CoA semialdehyde reductase kluyveri (orsuccinate semialdehyde dehydrogenase) sucD NP_904963.1 Porphyromonassuccinyl-CoA gingivalis Msed_0709 YP_001190808.1 Metallosphaeramalonyl-CoA sedula 8A 1.1.1.a succinate 4-hydroxy- 4-hydroxybutyrate4hbd YP_726053.1 Ralstonia 4-hydroxy- semialdehyde butyratedehydrogenase eutropha H16 butyrate 4hbd L21902.1 Clostridium 4-hydroxy-kluyveri DSM 555 butyrate 4hbd Q94B07 Arabidopsis 4-hydroxy- thalianabutyrate 8A 1.1.1.c succinyl-CoA 4-hydroxy- succinyl-CoA adhE2AAK09379.1 Clostridium butanoyl-CoA butyrate reductase acetobutylicum(alcohol forming) mcr AAS20429.1 Chloroflexus malonyl-CoA aurantiacusFAR AAD38039.1 Simmondsia long chain chinensis acyl-CoA 8A 2.8.3.a4-hydroxy- 4-hydroxy- 4-hydroxy- cat1, cat2, P38946.1, Clostridiumsuccinate, 4- butyrate butyryl-CoA butyryl-CoA cat3 P38942.2, kluyverihydroxybutyrate, transferase EDK35586.1 butyrate gctA, gctB CAA57199.1,Acidaminococcus glutarate CAA57200.1 fermentans atoA, atoD P76459.1,Escherichia butanoate P76458.1 coli 8A 3.1.2.a 4-hydroxy- 4-hydroxy-4-hydroxy- tesB NP_414986 Escherichia adipyl-CoA butyrate butyryl-CoAbutyryl-CoA coli hydrolase acot12 NP_570103.1 Rattus butyryl-CoAnorvegicus hibch Q6NVY1.2 Homo sapiens 3-hydroxy- propanoyl-CoA 8A6.2.1.a 4-hydroxy- 4-hydroxy- 4-hydroxy- sucCD NP_415256.1, Escherichiasuccinate butyrate butyryl-CoA butyryl-CoA AAC73823.1 coli ligase (or 4-hydroxybutyryl- CoA synthetase) phl CAJ15517.1 Penicillium phenylacetatechrysogenum bioW NP_390902.2 Bacillus 6-carboxy- subtilis hexanoate 8A2.7.2.a 4-hydroxy- 4-hydroxy- 4-hydroxy- ackA NP_416799.1 Escherichiaacetate, butyrate butyryl- butyrate coli propionate phosphate kinasebuk1 NP_349675 Clostridium butyrate acetobutylicum buk2 Q97II1Clostridium butyrate acetobutylicum 8A 2.3.1.a 4-hydroxy- 4-hydroxy-phosphotrans-4- ptb NP_349676 Clostridium butyryl- butyryl- butyryl-CoAhydroxybutyrylase acetobutylicum phosphate phosphate ptb AAR19757.1butyrate- butyryl- producing phosphate bacterium L2-50 ptb CAC07932.1Bacillus butyryl- megaterium phosphate 8A 1.2.1.d 4-hydroxy- 4-hydroxy-4-hydroxybutanal asd NP_417891.1 Escherichia L-4-aspartyl- butyryl-butanal dehydrogenase coli phosphate phosphate (phosphorylating) proANP_414778.1 Escherichia L-glutamyl- coli 5-phospate gapA P0A9B2.2Escherichia Glyceraldehyde- coli 3-phosphate 8A 1.2.1.b 4-hydroxy-4-hydroxy- 4-hydroxy- sucD P38947.1 Clostridium succinyl-CoA butyryl-CoAbutanal butyryl-CoA kluyveri reductase (or 4- hydroxybutanaldehydrogenase) sucD NP_904963.1 Porphyromonas succinyl-CoA gingivalisMsed_0709 YP_001190808.1 Metallosphaera malonyl-CoA sedula 8A 1.1.1.c4-hydroxy- 1,4- 4-hydroxy- adhE2 AAK09379.1 Clostridium butanoyl-CoAbutyryl-CoA butanediol butyryl-CoA acetobutylicum reductase (alcoholforming) mcr AAS20429.1 Chloroflexus malonyl-CoA aurantiacus FARAAD38039.1 Simmondsia long chain chinensis acyl-CoA 8A 1.1.1.a4-hydroxy- 1,4- 1,4-butanediol ADH2 NP_014032.1 Saccharymyces generalbutanal butanediol dehydrogenase cerevisiae yqhD NP_417484.1Escherichia >C3 coli 4hbd L21902.1 Clostridium Succinate kluyveri DSM555 semialdehyde

EXAMPLE VI Additional Exemplary BDO Pathways from Alpha-Ketoglutarate

This example describes exemplary BDO pathways from alpha-ketoglutarate.

BDO pathways from succinyl-CoA are described herein and have beendescribed previously (see U.S. application Ser. No. 12/049,256, filedMar. 14, 2008, and PCT application serial No. U.S.08/57168, filed Mar.14, 2008, each of which is incorporated herein by reference). Additionalpathways are shown in FIG. 8B. Enzymes of such exemplary BDO pathwaysare listed in Table 16, along with exemplary genes encoding theseenzymes.

Briefly, alpha-ketoglutarate can be converted to succinic semialdehydeby alpha-ketoglutarate decarboxylase (EC 4.1.1.a), as previouslydescribed. Alternatively, alpha-ketoglutarate can be converted toglutamate by glutamate dehydrogenase (EC 1.4.1.a). 4-Aminobutyrate canbe converted to succinic semialdehyde by 4-aminobutyrate oxidoreductase(deaminating) (EC 1.4.1.a) or 4-aminobutyrate transaminase (EC 2.6.1.a).Glutamate can be converted to 4-aminobutyrate by glutamate decarboxylase(EC 4.1.1.a). Succinate semialdehyde can be converted to4-hydroxybutyrate by 4-hydroxybutyrate dehydrogenase (EC 1.1.1.a), aspreviously described. 4-Hydroxybutyrate can be converted to4-hydroxybutyryl-CoA by 4-hydroxybutyryl-CoA transferase (EC 2.8.3.a),as previously described, or by 4-hydroxybutyryl-CoA hydrolase (EC3.1.2.a), or 4-hydroxybutyryl-CoA ligase (or 4-hydroxybutyryl-CoAsynthetase) (EC 6.2.1.a). 4-Hydroxybutyrate can be converted to4-hydroxybutyryl-phosphate by 4-hydroxybutyrate kinase (EC 2.7.2.a).4-Hydroxybutyryl-phosphate can be converted to 4-hydroxybutyryl-CoA byphosphotrans-4-hydroxybutyrylase (EC 2.3.1.a), as previously described.Alternatively, 4-hydroxybutyryl-phosphate can be converted to4-hydroxybutanal by 4-hydroxybutanal dehydrogenase (phosphorylating) (EC1.2.1.d). 4-Hydroxybutyryl-CoA can be converted to 4-hydroxybutanal by4-hydroxybutyryl-CoA reductase (or 4-hydroxybutanal dehydrogenase) (EC1.2.1.b), as previously described. 4-Hydroxybutyryl-CoA can be convertedto 1,4-butanediol by 4-hydroxybutyryl-CoA reductase (alcohol forming)(EC 1.1.1.c). 4-Hydroxybutanal can be converted to 1,4-butanediol by1,4-butanediol dehydrogenase (EC 1.1.1.a), as previously described.

TABLE 16 BDO pathway from alpha-ketoglutarate. EC Desired Desired GeneGenBank ID Known FIG. class substrate product Enzyme name name (ifavailable) Organism Substrates 8B 4.1.1.a alpha-keto- succinicalpha-keto- kgd O50463.4 Mycobacterium alpha-keto- glutaratesemialdehyde glutarate tuberculosis glutarate decarboxylase gadANP_417974 Escherichia glutamate coli gadB NP_416010 Escherichiaglutamate coli 8B 1.4.1.a alpha-keto- glutamate glutamate gdhA P00370Escherichia glutamate glutarate dehydrogenase coli gdh P96110.4Thermotoga glutamate maritima gdhA1 NP_279651.1 Halobacterium glutamatesalinarum 8B 1.4.1.a 4-amino- succinic 4-aminobutyrate lysDH AB052732Geobacillus lysine butyrate semialdehyde oxidoreductasestearothermophilus (deaminating) lysDH NP_147035.1 Aeropyrum lysinepernix K1 ldh P0A393 Bacillus leucine, cereus isoleucine, valine, 2-aminobutanoate 8B 2.6.1.a 4-amino- succinic 4-aminobutyrate gabTP22256.1 Escherichia 4-amino- butyrate semialdehyde transaminase colibutyryate puuE NP_415818.1 Escherichia 4-amino- coli butyryate UGA1NP_011533.1 Saccharomyces 4-amino- cerevisiae butyryate 8B 4.1.1.aglutamate 4-amino- glutamate gadA NP_417974 Escherichia glutamatebutyrate decarboxylase coli gadB NP_416010 Escherichia glutamate colikgd O50463.4 Mycobacterium alpha-keto- tuberculosis glutarate 8B 1.1.1.asuccinate 4-hydroxy- 4-hydroxybutyrate 4hbd YP_726053.1 Ralstonia4-hydroxy- semialdehyde butyrate dehydrogenase eutropha H16 butyrate4hbd L21902.1 Clostridium 4-hydroxy- kluyveri DSM 555 butyrate 4hbdQ94B07 Arabidopsis 4-hydroxy- thaliana butyrate 8B 2.8.3.a 4-hydroxy-4-hydroxy- 4-hydroxybutyryl- cat1, cat2, P38946.1, Clostridiumsuccinate, 4- butyrate butyryl-CoA CoA transferase cat3 P38942.2,kluyveri hydroxybutyrate, EDK35586.1 butyrate gctA, gctB CAA57199.1,Acidaminococcus glutarate CAA57200.1 fermentans atoA, atoD P76459.1,Escherichia butanoate P76458.1 coli 8B 3.1.2.a 4-hydroxy- 4-hydroxy-4-hydroxybutyryl- tesB NP_414986 Escherichia adipyl-CoA butyratebutyryl-CoA CoA hydrolase coli acot12 NP_570103.1 Rattus butyryl-CoAnorvegicus hibch Q6NVY1.2 Homo sapiens 3-hydroxy- propanoyl-CoA 8B6.2.1.a 4-hydroxy- 4-hydroxy- 4-hydroxybutyryl- sucCD NP_415256.1,Escherichia succinate butyrate butyryl-CoA CoA ligase (or 4- AAC73823.1coli hydroxybutyryl- CoA synthetase) phl CAJ15517.1 Penicilliumphenylacetate chrysogenum bioW NP_390902.2 Bacillus 6-carboxy- subtilishexanoate 8B 2.7.2.a 4-hydroxy- 4-hydroxy- 4-hydroxybutyrate ackANP_416799.1 Escherichia acetate, butyrate butyryl- kinase colipropionate phosphate buk1 NP_349675 Clostridium butyrate acetobutylicumbuk2 Q97II1 Clostridium butyrate acetobutylicum 8B 2.3.1.a 4-hydroxy-4-hydroxy- phosphotrans-4- ptb NP_349676 Clostridium butyryl- butyryl-butyryl-CoA hydroxybutyrylase acetobutylicum phosphate phosphate ptbAAR19757.1 butyrate- butyryl- producing phosphate bacterium L2-50 ptbCAC07932.1 Bacillus butyryl- megaterium phosphate 8B 1.2.1.d 4-hydroxy-4-hydroxy- 4-hydroxybutanal asd NP_417891.1 Escherichia L-4-aspartyl-butyryl- butanal dehydrogenase coli phosphate phosphate(phosphorylating) proA NP_414778.1 Escherichia L-glutamyl-5- coliphospate gapA P0A9B2.2 Escherichia Glyceraldehyde- coli 3-phosphate 8B1.2.1.b 4-hydroxy- 4-hydroxy- 4-hydroxybutyryl- sucD P38947.1Clostridium succinyl-CoA butyryl-CoA butanal CoA reductase (or kluyveri4-hydroxybutanal dehydrogenase) sucD NP_904963.1 Porphyromonassuccinyl-CoA gingivalis Msed_0709 YP_001190808.1 Metallosphaeramalonyl-CoA sedula 8B 1.1.1.c 4-hydroxy- 1,4- 4-hydroxybutyryl- adhE2AAK09379.1 Clostridium butanoyl-CoA butyryl-CoA butanediol CoA reductaseacetobutylicum (alcohol forming) mcr AAS20429.1 Chloroflexus malonyl-CoAaurantiacus FAR AAD38039.1 Simmondsia long chain chinensis acyl-CoA 8B1.1.1.a 4-hydroxy- 1,4- 1,4-butanediol ADH2 NP_014032.1 Saccharymycesgeneral butanal butanediol dehydrogenase cerevisiae yqhD NP_417484.1Escherichia >C3 coli 4hbd L21902.1 Clostridium Succinate kluyveri DSM555 semialdehyde

EXAMPLE VII BDO Pathways from 4-Aminobutyrate

This example describes exemplary BDO pathways from 4-aminobutyrate.

FIG. 9A depicts exemplary BDO pathways in which 4-aminobutyrate isconverted to BDO. Enzymes of such an exemplary BDO pathway are listed inTable 17, along with exemplary genes encoding these enzymes.

Briefly, 4-aminobutyrate can be converted to 4-aminobutyryl-CoA by4-aminobutyrate CoA transferase (EC 2.8.3.a), 4-aminobutyryl-CoAhydrolase (EC 3.1.2.a), or 4-aminobutyrate-CoA ligase (or4-aminobutyryl-CoA synthetase) (EC 6.2.1.a). 4-aminobutyryl-CoA can beconverted to 4-oxobutyryl-CoA by 4-aminobutyryl-CoA oxidoreductase(deaminating) (EC 1.4.1.a) or 4-aminobutyryl-CoA transaminase (EC2.6.1.a). 4-oxobutyryl-CoA can be converted to 4-hydroxybutyryl-CoA by4-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.a). 4-hydroxybutyryl-CoAcan be converted to 1,4-butanediol by 4-hydroxybutyryl-CoA reductase(alcohol forming) (EC 1.1.1.c). Alternatively, 4-hydroxybutyryl-CoA canbe converted to 4-hydroxybutanal by 4-hydroxybutyryl-CoA reductase (or4-hydroxybutanal dehydrogenase) (EC 1.2.1.b). 4-hydroxybutanal can beconverted to 1,4-butanediol by 1,4-butanediol dehydrogenase (EC1.1.1.a).

TABLE 17 BDO pathway from 4-aminobutyrate. EC Desired Desired GeneGenBank ID Known FIG. class substrate product Enzyme name name (ifavailable) Organism Substrates 9A 2.8.3.a 4-amino- 4-amino-4-aminobutyrate cat1, cat2, P38946.1, Clostridium succinate, 4- butyratebutyryl-CoA CoA transferase cat3 P38942.2, kluyveri hydroxybutyrate,EDK35586.1 butyrate gctA, gctB CAA57199.1, Acidaminococcus glutarateCAA57200.1 fermentans atoA, atoD P76459.1, Escherichia butanoateP76458.1 coli 9A 3.1.2.a 4-amino- 4-amino- 4-aminobutyryl- tesBNP_414986 Escherichia adipyl-CoA butyrate butyryl-CoA CoA hydrolase coliacot12 NP_570103.1 Rattus butyryl-CoA norvegicus hibch Q6NVY1.2 Homosapiens 3-hydroxy- propanoyl-CoA 9A 6.2.1.a 4-amino- 4-amino-4-aminobutyrate- sucCD NP_415256.1, Escherichia succinate butyratebutyryl-CoA CoA ligase (or 4- AAC73823.1 coli aminobutyryl-CoAsynthetase) phl CAJ15517.1 Penicillium phenylacetate chrysogenum bioWNP_390902.2 Bacillus 6-carboxy- subtilis hexanoate 9A 1.4.1.a 4-amino-4-oxo- 4-aminobutyryl- lysDH AB052732 Geobacillus lysine butyryl-CoAbutyryl-CoA CoA oxidoreductase stearothermophilus (deaminating) lysDHNP_147035.1 Aeropyrum lysine pernix K1 ldh P0A393 Bacillus leucine,isoleucine, cereus valine, 2- aminobutanoate 9A 2.6.1.a 4-amino- 4-oxo-4-aminobutyryl- gabT P22256.1 Escherichia 4-aminobutyryate butyryl-CoAbutyryl-CoA CoA transaminase coli abat P50554.3 Rattus 3-amino-2-norvegicus methylpropionate SkyPYD4 ABF58893.1 Saccharomycesbeta-alanine kluyveri 9A 1.1.1.a 4-oxo- 4-hydroxy- 4-hydroxybutyryl-ADH2 NP_014032.1 Saccharymyces general butyryl-CoA butyryl-CoA CoAdehydrogenase cerevisiae yqhD NP_417484.1 Escherichia >C3 coli 4hbdL21902.1 Clostridium Succinate kluyveri DSM 555 semialdehyde 8 1.1.1.c4-hydroxy- 1,4- 4-hydroxybutyryl- adhE2 AAK09379.1 Clostridiumbutanoyl-CoA butyryl-CoA butanediol CoA reductase acetobutylicum(alcohol forming) mcr AAS20429.1 Chloroflexus malonyl-CoA aurantiacusFAR AAD38039.1 Simmondsia long chain chinensis acyl-CoA 8 1.2.1.b4-hydroxy- 4-hydroxy- 4-hydroxybutyryl- sucD P38947.1 ClostridiumSuccinyl-CoA butyryl-CoA butanal CoA reductase (or kluyveri 4-hydroxybutanal dehydrogenase) sucD NP_904963.1 PorphyromonasSuccinyl-CoA gingivalis Msed_0709 YP_001190808.1 MetallosphaeraMalonyl-CoA sedula 8 1.1.1.a 4-hydroxy- 1,4- 1,4-butanediol ADH2NP_014032.1 Saccharymyces general butanal butanediol dehydrogenasecerevisiae yqhD NP_417484.1 Escherichia >C3 coli 4hbd L21902.1Clostridium Succinate kluyveri DSM 555 semialdehyde

Enzymes for another exemplary BDO pathway converting 4-aminobutyrate toBDO is shown in FIG. 9A. Enzymes of such an exemplary BDO pathway arelisted in Table 18, along with exemplary genes encoding these enzymes.

Briefly, 4-aminobutyrate can be converted to 4-aminobutyryl-CoA by4-aminobutyrate CoA transferase (EC 2.8.3.a), 4-aminobutyryl-CoAhydrolase (EC 3.1.2.a) or 4-aminobutyrate-CoA ligase (or4-aminobutyryl-CoA synthetase) (EC 6.2.1.a). 4-aminobutyryl-CoA can beconverted to 4-aminobutan-1-ol by 4-aminobutyryl-CoA reductase (alcoholforming) (EC 1.1.1.c). Alternatively, 4-aminobutyryl-CoA can beconverted to 4-aminobutanal by 4-aminobutyryl-CoA reductase (or4-aminobutanal dehydrogenase) (EC 1.2.1.b), and 4-aminobutanal convertedto 4-aminobutan-1-ol by 4-aminobutan-1-ol dehydrogenase (EC 1.1.1.a).4-aminobutan-1-ol can be converted to 4-hydroxybutanal by4-aminobutan-1-ol oxidoreductase (deaminating) (EC 1.4.1.a) or4-aminobutan-1-ol transaminase (EC 2.6.1.a). 4-hydroxybutanal can beconverted to 1,4-butanediol by 1,4-butanediol dehydrogenase (EC1.1.1.a).

TABLE 18 BDO pathway from 4-aminobutyrate. EC Desired Desired GeneGenBank ID Known FIG. class substrate product Enzyme name name (ifavailable) Organism Substrate 9A 2.8.3.a 4-amino- 4-amino-4-aminobutyrate cat1, cat2, P38946.1, Clostridium succinate, 4- butyratebutyryl-CoA CoA transferase cat3 P38942.2, kluyveri hydroxybutyrate,EDK35586.1 butyrate gctA, gctB CAA57199.1, Acidaminococcus glutarateCAA57200.1 fermentans atoA, atoD P76459.1, Escherichia butanoateP76458.1 coli 9A 3.1.2.a 4-amino- 4-amino- 4-aminobutyryl- tesBNP_414986 Escherichia adipyl-CoA butyrate butyryl-CoA CoA hydrolase coliacot12 NP_570103.1 Rattus butyryl-CoA norvegicus hibch Q6NVY1.2 Homosapiens 3-hydroxy- propanoyl-CoA 9A 6.2.1.a 4-amino- 4-amino-4-aminobutyrate- sucCD NP_415256.1, Escherichia succinate butyratebutyryl-CoA CoA ligase (or 4- AAC73823.1 coli aminobutyryl-CoAsynthetase) phl CAJ15517.1 Penicillium phenylacetate chrysogenum bioWNP_390902.2 Bacillus 6-carboxy- subtilis hexanoate 9A 1.1.1.c 4-amino-4-amino- 4-aminobutyryl- adhE2 AAK09379.1 Clostridium butanoyl-CoAbutyryl-CoA butan-1-ol CoA reductase acetobutylicum (alcohol forming)mcr AAS20429.1 Chloroflexus malonyl-CoA aurantiacus FAR AAD38039.1Simmondsia long chain chinensis acyl-CoA 9A 1.2.1.b 4-amino- 4-amino-4-aminobutyryl- sucD P38947.1 Clostridium Succinyl-CoA butyryl-CoAbutanal CoA reductase (or kluyveri 4-aminobutanal dehydrogenase) sucDNP_904963.1 Porphyromonas Succinyl-CoA gingivalis Msed_0709YP_001190808.1 Metallosphaera Malonyl-CoA sedula 9A 1.1.1.a 4-amino-4-amino- 4-aminobutan-1-ol ADH2 NP_014032.1 Saccharymyces generalbutanal butan-1-ol dehydrogenase cerevisiae yqhD NP_417484.1Escherichia >C3 coli 4hbd L21902.1 Clostridium Succinate kluyveri DSM555 semialdehyde 9A 1.4.1.a 4-amino- 4-hydroxy- 4-aminobutan-1-ol lysDHAB052732 Geobacillus lysine butan-1-ol butanal oxidoreductasestearothermophilus (deaminating) lysDH NP_147035.1 Aeropyrum lysinepernix K1 ldh P0A393 Bacillus leucine, cereus isoleucine, valine, 2-aminobutanoate 9A 2.6.1.a 4-amino- 4-hydroxy- 4-aminobutan-1-ol gabTP22256.1 Escherichia 4-amino- butan-1-ol butanal transaminase colibutyryate abat P50554.3 Rattus 3-amino-2- norvegicus methylpropionateSkyPYD4 ABF58893.1 Saccharomyces beta-alanine kluyveri 9A 1.1.1.a4-hydroxy- 1,4- 1,4-butanediol ADH2 NP_014032.1 Saccharymyces generalbutanal butanediol dehydrogenase cerevisiae yqhD NP_417484.1Escherichia >C3 coli 4hbd L21902.1 Clostridium Succinate kluyveri DSM555 semialdehyde

FIG. 9B depicts exemplary BDO pathway in which 4-aminobutyrate isconverted to BDO. Enzymes of such an exemplary BDO pathway are listed inTable 19, along with exemplary genes encoding these enzymes.

Briefly, 4-aminobutyrate can be converted to[(4-aminobutanolyl)oxy]phosphonic acid by 4-aminobutyrate kinase (EC2.7.2.a). [(4-aminobutanolyl)oxy]phosphonic acid can be converted to4-aminobutanal by 4-aminobutyraldehyde dehydrogenase (phosphorylating)(EC 1.2.1.d). 4-aminobutanal can be converted to 4-aminobutan-1-ol by4-aminobutan-1-ol dehydrogenase (EC 1.1.1.a). 4-aminobutan-1-ol can beconverted to 4-hydroxybutanal by 4-aminobutan-1-ol oxidoreductase(deaminating) (EC 1.4.1.a) or 4-aminobutan-1-ol transaminase (EC2.6.1.a). Alternatively, [(4-aminobutanolyl)oxy]phosphonic acid can beconverted to [(4-oxobutanolyl)oxy]phosphonic acid by[(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase (deaminating) (EC1.4.1.a) or [(4-aminobutanolyl)oxy]phosphonic acid transaminase (EC2.6.1.a). [(4-oxobutanolyl)oxy]phosphonic acid can be converted to4-hydroxybutyryl-phosphate by 4-hydroxybutyryl-phosphate dehydrogenase(EC 1.1.1.a). 4-hydroxybutyryl-phosphate can be converted to4-hydroxybutanal by 4-hydroxybutyraldehyde dehydrogenase(phosphorylating) (EC 1.2.1.d). 4-hydroxybutanal can be converted to1,4-butanediol by 1,4-butanediol dehydrogenase (EC 1.1.1.a).

TABLE 19 BDO pathway from 4-aminobutyrate. EC Desired Desired GeneGenBank ID Known FIG. class substrate product Enzyme name name (ifavailable) Organism Substrate 9B 2.7.2.a 4-amino- [(4-amino- 4-amino-ackA NP_416799.1 Escherichia acetate, butyrate butanolyl)oxy] butyratekinase coli propionate phosphonic acid buk1 NP_349675 Clostridiumbutyrate acetobutylicum proB NP_414777.1 Escherichia glutamate coli 9B1.2.1.d [(4-amino- 4-amino- 4-amino- asd NP_417891.1 EscherichiaL-4-aspartyl- butanolyl)oxy] butanal butyraldehyde coli phosphatephosphonic dehydrogenase acid (phosphorylating) proA NP_414778.1Escherichia L-glutamyl- coli 5-phospate gapA P0A9B2.2 EscherichiaGlyceraldehyde- coli 3-phosphate 9B 1.1.1.a 4-amino- 4-amino-4-aminobutan-1-ol ADH2 NP_014032.1 Saccharymyces general butanalbutan-1-ol dehydrogenase cerevisiae yqhD NP_417484.1 Escherichia >C3coli 4hbd L21902.1 Clostridium Succinate kluyveri DSM 555 semialdehyde9B 1.4.1.a 4-amino- 4-hydroxy- 4-aminobutan-1-ol lysDH AB052732Geobacillus lysine butan-1-ol butanal oxidoreductase stearothermophilus(deaminating) lysDH NP_147035.1 Aeropyrum lysine pernix K1 ldh P0A393Bacillus leucine, cereus isoleucine, valine, 2-aminobutanoate 9B 2.6.1.a4-amino- 4-hydroxy- 4-aminobutan-1-ol gabT P22256.1 Escherichia4-aminobutyryate butan-1-ol butanal transaminase coli abat P50554.3Rattus 3-amino-2- norvegicus methylpropionate SkyPYD4 ABF58893.1Saccharomyces beta-alanine kluyveri 9B 1.4.1.a [(4-amino- [(4-oxo-[(4-amino- lysDH AB052732 Geobacillus lysine butanolyl)oxy]butanolyl)oxy] butanolyl)oxy]phos- stearothermophilus phosphonicphosphonic phonic acid acid acid oxidoreductase (deaminating) lysDHNP_147035.1 Aeropyrum lysine pernix K1 ldh P0A393 Bacillus leucine,cereus isoleucine, valine, 2-aminobutanoate 9B 2.6.1.a [(4-amino-[(4-oxo- [(4-amino- gabT P22256.1 Escherichia 4-aminobutyryatebutanolyl)oxy] butanolyl)oxy] butanolyl)oxy]phos- coli phosphonicphosphonic phonic acid acid acid transaminase SkyPYD4 ABF58893.1Saccharomyces beta-alanine kluyveri serC NP_415427.1 Escherichiaphosphoserine, coli phospho- hydroxythreonine 9B 1.1.1.a [(4-oxo-4-hydroxy- 4-hydroxy- ADH2 NP_014032.1 Saccharymyces generalbutanolyl)oxy] butyryl- butyryl- cerevisiae phosphonic phosphatephosphate acid dehydrogenase yqhD NP_417484.1 Escherichia >C3 coli 4hbdL21902.1 Clostridium Succinate kluyveri DSM 555 semialdehyde 9B 1.2.1.d4-hydroxy- 4-hydroxy- 4-hydroxy- asd NP_417891.1 EscherichiaL-4-aspartyl- butyryl- butanal butyraldehyde coli phosphate phosphatedehydrogenase (phosphorylating) proA NP_414778.1 Escherichia L-glutamyl-coli 5-phospate gapA P0A9B2.2 Escherichia Glyceraldehyde- coli3-phosphate 9B 1.1.1.a 4-hydroxy- 1,4- 1,4-butanediol ADH2 NP_014032.1Saccharymyces general butanal butanediol dehydrogenase cerevisiae yqhDNP_417484.1 Escherichia >C3 coli 4hbd L21902.1 Clostridium Succinatekluyveri DSM 555 semialdehyde

FIG. 9C shows an exemplary pathway through acetoacetate.

EXAMPLE VIII Exemplary BDO Pathways from Alpha-Ketoglutarate

This example describes exemplary BDO pathways from alpha-ketoglutarate.

FIG. 10 depicts exemplary BDO pathways in which alpha-ketoglutarate isconverted to BDO. Enzymes of such an exemplary BDO pathway are listed inTable 20, along with exemplary genes encoding these enzymes.

Briefly, alpha-ketoglutarate can be converted toalpha-ketoglutaryl-phosphate by alpha-ketoglutarate 5-kinase (EC2.7.2.a). Alpha-ketoglutaryl-phosphate can be converted to2,5-dioxopentanoic acid by 2,5-dioxopentanoic semialdehyde dehydrogenase(phosphorylating) (EC 1.2.1.d). 2,5-dioxopentanoic acid can be convertedto 5-hydroxy-2-oxopentanoic acid by 2,5-dioxopentanoic acid reductase(EC 1.1.1.a). Alternatively, alpha-ketoglutarate can be converted toalpha-ketoglutaryl-CoA by alpha-ketoglutarate CoA transferase (EC2.8.3.a), alpha-ketoglutaryl-CoA hydrolase (EC 3.1.2.a) oralpha-ketoglutaryl-CoA ligase (or alpha-ketoglutaryl-CoA synthetase) (EC6.2.1.a). Alpha-ketoglutaryl-CoA can be converted to 2,5-dioxopentanoicacid by alpha-ketoglutaryl-CoA reductase (or 2,5-dioxopentanoic aciddehydrogenase) (EC 1.2.1.b). 2,5-Dioxopentanoic acid can be converted to5-hydroxy-2-oxopentanoic acid by 5-hydroxy-2-oxopentanoic aciddehydrogenase. Alternatively, alpha-ketoglutaryl-CoA can be converted to5-hydroxy-2-oxopentanoic acid by alpha-ketoglutaryl-CoA reductase(alcohol forming) (EC 1.1.1.c). 5-hydroxy-2-oxopentanoic acid can beconverted to 4-hydroxybutanal by 5-hydroxy-2-oxopentanoic aciddecarboxylase (EC 4.1.1.a). 4-hydroxybutanal can be converted to1,4-butanediol by 1,4-butanediol dehydrogenase (EC 1.1.1.a).5-hydroxy-2-oxopentanoic acid can be converted to 4-hydroxybutyryl-CoAby 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation) (EC1.2.1.c).

TABLE 20 BDO pathway from alpha-ketoglutarate. EC Desired Desired GeneGenBank ID Known FIG. class substrate product Enzyme name name (ifavailable) Organism Substrate 10 2.7.2.a alpha-keto- alpha-keto-alpha-keto- ackA NP_416799.1 Escherichia acetate, glutarate glutaryl-glutarate coli propionate phosphate 5-kinase buk1 NP_349675 Clostridiumbutyrate acetobutylicum proB NP_414777.1 Escherichia glutamate coli 101.2.1.d alpha-keto- 2,5-dioxo- 2,5- proA NP_414778.1 EscherichiaL-glutamyl- glutaryl- pentanoic acid dioxopentanoic coli 5-phospatephosphate semialdehyde dehydrogenase (phosphorylating) asd NP_417891.1Escherichia L-4-aspartyl- coli phosphate gapA P0A9B2.2 EscherichiaGlyceraldehyde- coli 3-phosphate 10 1.1.1.a 2,5-dioxo- 5-hydroxy-2- 2,5-ADH2 NP_014032.1 Saccharymyces general pentanoic acid oxopentanoicdioxopentanoic cerevisiae acid acid reductase yqhD NP_417484.1Escherichia >C3 coli 4hbd L21902.1 Clostridium Succinate kluyveri DSM555 semialdehyde 10 2.8.3.a alpha-keto- alpha-keto- alpha-keto- cat1,cat2, P38946.1, Clostridium succinate, 4- glutarate glutaryl-CoAglutarate CoA cat3 P38942.2, kluyveri hydroxybutyrate, transferaseEDK35586.1 butyrate gctA, gctB CAA57199.1, Acidaminococcus glutarateCAA57200.1 fermentans atoA, atoD P76459.1, Escherichia butanoateP76458.1 coli 10 3.1.2.a alpha-keto- alpha-keto- alpha-keto- tesBNP_414986 Escherichia adipyl-CoA glutarate glutaryl-CoA glutaryl-CoAcoli hydrolase acot12 NP_570103.1 Rattus butyryl-CoA norvegicus hibchQ6NVY1.2 Homo sapiens 3-hydroxy- propanoyl-CoA 10 6.2.1.a alpha-keto-alpha-keto- alpha- sucCD NP_415256.1, Escherichia succinate glutarateglutaryl-CoA ketoglutaryl- AAC73823.1 coli CoA ligase (or alpha-ketoglutaryl- CoA synthetase) phl CAJ15517.1 Penicillium phenylacetatechrysogenum bioW NP_390902.2 Bacillus 6-carboxy- subtilis hexanoate 101.2.1.b alpha-keto- 2,5-dioxo- alpha- sucD P38947.1 ClostridiumSuccinyl-CoA glutaryl-CoA pentanoic acid ketoglutaryl- kluyveri CoAreductase (or 2,5- dioxopentanoic acid dehydrogenase) Msed_0709YP_001190808.1 Metallosphaera Malonyl-CoA sedula bphG BAA03892.1Pseudomonas sp Acetaldehyde, Propionaldehyde, Butyraldehyde,Isobutyraldehyde and Formaldehyde 10 1.1.1.a 2,5-dioxo- 5-hydroxy-2-5-hydroxy-2- ADH2 NP_014032.1 Saccharymyces general pentanoic acidoxopentanoic oxopentanoic yqhD NP_417484.1 cerevisiae >C3 acid acid 4hbdL21902.1 Escherichia Succinate dehydrogenase coli semialdehydeClostridium kluyveri DSM 555 10 1.1.1.c alpha-keto- 5-hydroxy-2- alpha-adhE2 AAK09379.1 Clostridium butanoyl-CoA glutaryl-CoA oxopentanoicketoglutaryl- acetobutylicum acid CoA reductase (alcohol forming) mcrAAS20429.1 Chloroflexus malonyl-CoA aurantiacus FAR AAD38039.1Simmondsia long chain chinensis acyl-CoA 10 4.1.1.a 5-hydroxy-2-4-hydroxy- 5-hydroxy-2- pdc P06672.1 Zymomonas 2-oxopentanoicoxopentanoic butanal oxopentanoic mobilus acid acid acid decarboxylasemdlC P20906.2 Pseudomonas 2-oxopentanoic putida acid pdc1 P06169Saccharomyces pyruvate cerevisiae 10 1.1.1.a 4-hydroxy- 1,4-1,4-butanediol ADH2 NP_014032.1 Saccharymyces general butanal butanedioldehydrogenase cerevisiae yqhD NP_417484.1 Escherichia >C3 coli 4hbdL21902.1 Clostridium Succinate kluyveri DSM 555 semialdehyde 10 1.2.1.c5-hydroxy-2- 4-hydroxy- 5-hydroxy-2- sucA, sucB, NP_415254.1,Escherichia Alpha- oxopentanoic butyryl-CoA oxopentanoic lpdNP_415255.1, coli ketoglutarate acid acid NP_414658.1 dehydrogenase(decarboxylation) bfmBB, NP_390283.1, Bacillus 2-keto acids bfmBAA,NP_390285.1, subtilis derivatives of bfmBAB, NP_390284.1, valine,leucine bfmBAB, P21880.1 and isoleucine pdhD Bckdha, NP_036914.1, Rattus2-keto acids Bckdhb, NP_062140.1, norvegicus derivatives of Dbt, DldNP_445764.1, valine, leucine NP_955417.1 and isoleucine

EXAMPLE IX Exemplary BDO Pathways from Glutamate

This example describes exemplary BDO pathways from glutamate.

FIG. 11 depicts exemplary BDO pathways in which glutamate is convertedto BDO. Enzymes of such an exemplary BDO pathway are listed in Table 21,along with exemplary genes encoding these enzymes.

Briefly, glutamate can be converted to glutamyl-CoA by glutamate CoAtransferase (EC 2.8.3.a), glutamyl-CoA hydrolase (EC 3.1.2.a) orglutamyl-CoA ligase (or glutamyl-CoA synthetase) (EC 6.2.1.a).Alternatively, glutamate can be converted to glutamate-5-phosphate byglutamate 5-kinase (EC 2.7.2.a). Glutamate-5-phosphate can be convertedto glutamate-5-semialdehyde by glutamate-5-semialdehyde dehydrogenase(phosphorylating) (EC 1.2.1.d). Glutamyl-CoA can be converted toglutamate-5-semialdehyde by glutamyl-CoA reductase (orglutamate-5-semialdehyde dehydrogenase) (EC 1.2.1.b).Glutamate-5-semialdehyde can be converted to 2-amino-5-hydroxypentanoicacid by glutamate-5-semialdehyde reductase (EC 1.1.1.a). Alternatively,glutamyl-CoA can be converted to 2-amino-5-hydroxypentanoic acid byglutamyl-CoA reductase (alcohol forming) (EC 1.1.1.c).2-Amino-5-hydroxypentanoic acid can be converted to5-hydroxy-2-oxopentanoic acid by 2-amino-5-hydroxypentanoic acidoxidoreductase (deaminating) (EC 1.4.1.a) or 2-amino-5-hydroxypentanoicacid transaminase (EC 2.6.1.a). 5-Hydroxy-2-oxopentanoic acid can beconverted to 4-hydroxybutanal by 5-hydroxy-2-oxopentanoic aciddecarboxylase (EC 4.1.1.a). 4-Hydroxybutanal can be converted to1,4-butanediol by 1,4-butanediol dehydrogenase (EC 1.1.1.a).Alternatively, 5-hydroxy-2-oxopentanoic acid can be converted to4-hydroxybutyryl-CoA by 5-hydroxy-2-oxopentanoic acid dehydrogenase(decarboxylation) (EC 1.2.1.c).

TABLE 21 BDO pathway from glutamate. EC Desired Desired Gene GenBank IDKnown FIG. class substrate product Enzyme name name (if available)Organism Substrate 11 2.8.3.a glutamate glutamyl- glutamate CoA cat1,cat2, P38946.1, Clostridium succinate, 4- CoA transferase cat3 P38942.2,kluyveri hydroxybutyrate, EDK35586.1 butyrate gctA, gctB CAA57199.1,Acidaminococcus glutarate CAA57200.1 fermentans atoA, atoD P76459.1,Escherichia butanoate P76458.1 coli 11 3.1.2.a glutamate glutamyl-glutamyl-CoA tesB NP_414986 Escherichia adipyl-CoA CoA hydrolase coliacot12 NP_570103.1 Rattus butyryl-CoA norvegicus hibch Q6NVY1.2 Homosapiens 3-hydroxy- propanoyl-CoA 11 6.2.1.a glutamate glutamyl-glutamyl-CoA sucCD NP_415256.1, Escherichia succinate CoA ligase (orAAC73823.1 coli glutamyl- CoA synthetase) phl CAJ15517.1 Penicilliumphenylacetate chrysogenum bioW NP_390902.2 Bacillus 6-carboxy- subtilishexanoate 11 2.7.2.a glutamate glutamate-5- glutamate ackA NP_416799.1Escherichia acetate, phosphate 5-kinase coli propionate buk1 NP_349675Clostridium butyrate acetobutylicum proB NP_414777.1 Escherichiaglutamate coli 11 1.2.1.d glutamate-5- glutamate- glutamate-5- proANP_414778.1 Escherichia L-glutamyl-5- phosphate 5-semi- semialdehydecoli phospate aldehyde dehydrogenase (phosphorylating) asd NP_417891.1Escherichia L-4-aspartyl- coli phosphate gapA P0A9B2.2 EscherichiaGlyceraldehyde- coli 3-phosphate 11 1.2.1.b glutamyl- glutamate-glutamyl-CoA sucD P38947.1 Clostridium Succinyl-CoA CoA 5-semi-reductase (or kluyveri aldehyde glutamate-5- semialdehyde dehydrogenase)Msed_0709 YP_001190808.1 Metallosphaera Malonyl-CoA sedula bphGBAA03892.1 Pseudomonas sp Acetaldehyde, Propionaldehyde, Butyraldehyde,Isobutyraldehyde and Formaldehyde 11 1.1.1.a glutamate- 2-amino-5-glutamate-5- ADH2 NP_014032.1 Saccharymyces general 5-semi- hydroxy-semialdehyde cerevisiae aldehyde pentanoic reductase acid yqhDNP_417484.1 Escherichia >C3 coli 4hbd L21902.1 Clostridium Succinatekluyveri DSM 555 semialdehyde 11 1.1.1.c glutamyl- 2-amino-5-glutamyl-CoA adhE2 AAK09379.1 Clostridium butanoyl-CoA CoA hydroxy-reductase acetobutylicum pentanoic (alcohol forming) acid mcr AAS20429.1Chloroflexus malonyl-CoA aurantiacus FAR AAD38039.1 Simmondsia longchain chinensis acyl-CoA 11 1.4.1.a 2-amino-5- 5-hydroxy-2- 2-amino-5-gdhA P00370 Escherichia glutamate hydroxy- oxopentanoic hydroxypentanoiccoli pentanoic acid acid oxido- acid reductase (deaminating) ldh P0A393Bacillus leucine, cereus isoleucine, valine, 2- aminobutanoate nadXNP_229443.1 Thermotoga aspartate maritima 11 2.6.1.a 2-amino-5-5-hydroxy-2- 2-amino-5- aspC NP_415448.1 Escherichia aspartate hydroxy-oxopentanoic hydroxy- coli pentanoic acid pentanoic acid acidtransaminase AAT2 P23542.3 Saccharomyces aspartate cerevisiae avtAYP_026231.1 Escherichia valine, alpha- coli aminobutyrate 11 4.1.1.a5-hydroxy-2- 4-hydroxy- 5-hydroxy-2- pdc P06672.1 Zymomonas2-oxopentanoic oxopentanoic butanal oxopentanoic acid mobilus acid aciddecarboxylase mdlC P20906.2 Pseudomonas 2-oxopentanoic putida acid pdc1P06169 Saccharomyces pyruvate cerevisiae 11 1.1.1.a 4-hydroxy- 1,4-1,4-butanediol ADH2 NP_014032.1 Saccharymyces general butanal butanedioldehydrogenase cerevisiae yqhD NP_417484.1 Escherichia >C3 coli 4hbdL21902.1 Clostridium Succinate kluyveri DSM 555 semialdehyde 11 1.2.1.c5-hydroxy-2- 4-hydroxy- 5-hydroxy-2- sucA, sucB, NP_415254.1,Escherichia Alpha-keto- oxopentanoic butyryl-CoA oxopentanoic acid lpdNP_415255.1, coli glutarate acid dehydrogenase NP_414658.1(decarboxylation) bfmBB, NP_390283.1, Bacillus 2-keto acids bfmBAA,NP_390285.1, subtilis derivatives of bfmBAB, NP_390284.1, valine,leucine bfmBAB, pdhD P21880.1 and isoleucine Bckdha, NP_036914.1, Rattus2-keto acids Bckdhb, NP_062140.1, norvegicus derivatives of Dbt, DldNP_445764.1, valine, leucine NP_955417.1 and isoleucine

EXAMPLE X Exemplary BDO from Acetoacetyl-CoA

This example describes an exemplary BDO pathway from acetoacetyl-CoA.

FIG. 12 depicts exemplary BDO pathways in which acetoacetyl-CoA isconverted to BDO. Enzymes of such an exemplary BDO pathway are listed inTable 22, along with exemplary genes encoding these enzymes.

Briefly, acetoacetyl-CoA can be converted to 3-hydroxybutyryl-CoA by3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.a). 3-Hydroxybutyryl-CoAcan be converted to crotonoyl-CoA by 3-hydroxybutyryl-CoA dehydratase(EC 4.2.1.a). Crotonoyl-CoA can be converted to vinylacetyl-CoA byvinylacetyl-CoA Δ-isomerase (EC 5.3.3.3). Vinylacetyl-CoA can beconverted to 4-hydroxybutyryl-CoA by 4-hydroxybutyryl-CoA dehydratase(EC 4.2.1.a). 4-Hydroxybutyryl-CoA can be converted to 1,4-butanediol by4-hydroxybutyryl-CoA reductase (alcohol forming) (EC 1.1.1.c).Alternatively, 4-hydroxybutyryl-CoA can be converted to 4-hydroxybutanalby 4-hydroxybutyryl-CoA reductase (or 4-hydroxybutanal dehydrogenase)(EC 1.2.1.b). 4-Hydroxybutanal can be converted to 1,4-butanediol by1,4-butanediol dehydrogenase (EC 1.1.1.a).

TABLE 22 BDO pathway from acetoacetyl-CoA. EC Desired Desired GeneGenBank ID Known FIG. class substrate product Enzyme name name (ifavailable) Organism Substrate 12 1.1.1.a acetoacetyl- 3-hydroxy-3-hydroxy- hbd NP_349314.1 Clostridium 3-hydroxy- CoA butyryl-CoAbutyryl-CoA acetobutylicum butyryl-CoA dehydrogenase hbd AAM14586.1Clostridium 3-hydroxy- beijerinckii butyryl-CoA Msed_1423 YP_001191505Metallosphaera presumed 3- sedula hydroxy- butyryl-CoA 12 4.2.1.a3-hydroxy- crotonoyl- 3-hydroxy- crt NP_349318.1 Clostridium 3-hydroxy-butyryl-CoA CoA butyryl-CoA acetobutylicum butyryl-CoA dehydratase maoCNP_415905.1 Escherichia 3-hydroxy- coli butyryl-CoA paaF NP_415911.1Escherichia 3-hydroxy- coli adipyl-CoA 12 5.3.3.3 crotonoyl-vinylacetyl- vinylacetyl- abfD YP_001396399.1 Clostridium 4-hydroxy- CoACoA CoA Δ- kluyveri DSM 555 butyryl-CoA isomerase abfD P55792Clostridium 4-hydroxy- aminobutyricum butyryl-CoA abfD YP_001928843Porphyromonas 4-hydroxy- gingivalis butyryl-CoA ATCC 33277 12 4.2.1.avinylacetyl- 4-hydroxy- 4-hydroxy- abfD YP_001396399.1 Clostridium4-hydroxy- CoA butyryl-CoA butyryl-CoA kluyveri DSM 555 butyryl-CoAdehydratase abfD P55792 Clostridium 4-hydroxy- aminobutyricumbutyryl-CoA abfD YP_001928843 Porphyromonas 4-hydroxy- gingivalisbutyryl-CoA ATCC 33277 12 1.1.1.c 4-hydroxy- 1,4- 4-hydroxy- adhE2AAK09379.1 Clostridium butanoyl-CoA butyryl-CoA butanediol butyryl-CoAacetobutylicum reductase (alcohol forming) mcr AAS20429.1 Chloroflexusmalonyl-CoA aurantiacus FAR AAD38039.1 Simmondsia long chain chinensisacyl-CoA 12 1.2.1.b 4-hydroxy- 4-hydroxy- 4-hydroxy- sucD P38947.1Clostridium Succinyl-CoA butyryl-CoA butanal butyryl-CoA kluyverireductase (or 4-hydroxy- butanal dehydrogenase) sucD NP_904963.1Porphyromonas Succinyl-CoA gingivalis Msed_0709 YP_001190808.1Metallosphaera Malonyl-CoA sedula 12 1.1.1.a 4-hydroxy- 1,4- 1,4- ADH2NP_014032.1 Saccharymyces general butanal butanediol butanediolcerevisiae dehydrogenase yqhD NP_417484.1 Escherichia >C3 coli 4hbdL21902.1 Clostridium Succinate kluyveri DSM 555 semialdehyde

EXAMPLE XI Exemplary BDO Pathway from Homoserine

This example describes an exemplary BDO pathway from homoserine.

FIG. 13 depicts exemplary BDO pathways in which homoserine is convertedto BDO. Enzymes of such an exemplary BDO pathway are listed in Table 23,along with exemplary genes encoding these enzymes.

Briefly, homoserine can be converted to 4-hydroxybut-2-enoate byhomoserine deaminase (EC 4.3.1.a). Alternatively, homoserine can beconverted to homoserine-CoA by homoserine CoA transferase (EC 2.8.3.a),homoserine-CoA hydrolase (EC 3.1.2.a) or homoserine-CoA ligase (orhomoserine-CoA synthetase) (EC 6.2.1.a). Homoserine-CoA can be convertedto 4-hydroxybut-2-enoyl-CoA by homoserine-CoA deaminase (EC 4.3.1.a).4-Hydroxybut-2-enoate can be converted to 4-hydroxybut-2-enoyl-CoA by4-hydroxybut-2-enoyl-CoA transferase (EC 2.8.3.a),4-hydroxybut-2-enoyl-CoA hydrolase (EC 3.1.2.a), or4-hydroxybut-2-enoyl-CoA ligase (or 4-hydroxybut-2-enoyl-CoA synthetase)(EC 6.2.1.a). Alternatively, 4-hydroxybut-2-enoate can be converted to4-hydroxybutyrate by 4-hydroxybut-2-enoate reductase (EC 1.3.1.a).4-Hydroxybutyrate can be converted to 4-hydroxybutyryl-coA by4-hydroxybutyryl-CoA transferase (EC 2.8.3.a), 4-hydroxybutyryl-CoAhydrolase (EC 3.1.2.a), or 4-hydroxybutyryl-CoA ligase (or4-hydroxybutyryl-CoA synthetase) (EC 6.2.1.a). 4-Hydroxybut-2-enoyl-CoAcan be converted to 4-hydroxybutyryl-CoA by 4-hydroxybut-2-enoyl-CoAreductase (EC 1.3.1.a). 4-Hydroxybutyryl-CoA can be converted to1,4-butanediol by 4-hydroxybutyryl-CoA reductase (alcohol forming) (EC1.1.1.c). Alternatively, 4-hydroxybutyryl-CoA can be converted to4-hydroxybutanal by 4-hydroxybutyryl-CoA reductase (or 4-hydroxybutanaldehydrogenase) (EC 1.2.1.b). 4-Hydroxybutanal can be converted to1,4-butanediol by 1,4-butanediol dehydrogenase (EC 1.1.1.a).

TABLE 23 BDO pathway from homoserine. EC Desired Desired Gene GenBank IDKnown FIG. class substrate product Enzyme name name (if available)Organism Substrate 13 4.3.1.a homoserine 4-hydroxybut- homoserine aspANP_418562 Escherichia aspartate 2-enoate deaminase coli aspA P44324.1Haemophilus aspartate influenzae aspA P07346 Pseudomonas aspartatefluorescens 13 2.8.3.a homoserine homoserine- homoserine CoA cat1, cat2,P38946.1, Clostridium succinate, 4- CoA transferase cat3 P38942.2,kluyveri hydroxybutyrate, EDK35586.1 butyrate gctA, gctB CAA57199.1,Acidaminococcus glutarate CAA57200.1 fermentans atoA, atoD P76459.1,Escherichia butanoate P76458.1 coli 13 3.1.2.a homoserine homoserine-homoserine-CoA tesB NP_414986 Escherichia adipyl-CoA CoA hydrolase coliacot12 NP_570103.1 Rattus butyryl-CoA norvegicus hibch Q6NVY1.2 Homosapiens 3-hydroxy- propanoyl-CoA 13 6.2.1.a homoserine homoserine-homoserine-CoA sucCD NP_415256.1, Escherichia succinate CoA ligase (orAAC73823.1 coli homoserine-CoA synthetase) phl CAJ15517.1 Penicilliumphenylacetate chrysogenum bioW NP_390902.2 Bacillus 6-carboxy- subtilishexanoate 13 4.3.1.a homoserine- 4-hydroxybut- homoserine-CoA acl1CAG29274.1 Clostridium beta-alanyl- CoA 2-enoyl-CoA deaminasepropionicum CoA acl2 CAG29275.1 Clostridium beta-alanyl- propionicum CoAMXAN_4385 YP_632558.1 Myxococcus beta-alanyl- xanthus CoA 13 2.8.3.a4-hydroxybut- 4-hydroxybut- 4-hydroxybut-2- cat1, cat2, P38946.1,Clostridium succinate, 4- 2-enoate 2-enoyl-CoA enoyl-CoA cat3 P38942.2,kluyveri hydroxybutyrate, transferase EDK35586.1 butyrate gctA, gctBCAA57199.1, Acidaminococcus glutarate CAA57200.1 fermentans atoA, atoDP76459.1, Escherichia butanoate P76458.1 coli 13 3.1.2.a 4-hydroxybut-4-hydroxybut- 4-hydroxybut-2- tesB NP_414986 Escherichia adipyl-CoA2-enoate 2-enoyl-CoA enoyl-CoA coli hydrolase acot12 NP_570103.1 Rattusbutyryl-CoA norvegicus hibch Q6NVY1.2 Homo sapiens 3-hydroxy-propanoyl-CoA 13 6.2.1.a 4-hydroxybut- 4-hydroxybut- 4-hydroxybut-2-sucCD NP_415256.1, Escherichia succinate 2-enoate 2-enoyl-CoA enoyl-CoAAAC73823.1 coli ligase (or 4- hydroxybut-2- enoyl-CoA synthetase) phlCAJ15517.1 Penicillium phenylacetate chrysogenum bioW NP_390902.2Bacillus 6-carboxy- subtilis hexanoate 13 1.3.1.a 4-hydroxybut-4-hydroxy- 4-hydroxybut-2- enr CAA71086.1 Clostridium 2-enoate butyrateenoate reductase tyrobutyricum enr CAA76083.1 Clostridium kluyveri enrYP_430895.1 Moorella thermoacetica 13 2.8.3.a 4-hydroxy- 4-hydroxy-4-hydroxybutyryl- cat1, cat2, P38946.1, Clostridium succinate, 4-butyrate butyryl-coA CoA transferase cat3 P38942.2, kluyverihydroxybutyrate, EDK35586.1 butyrate gctA, gctB CAA57199.1,Acidaminococcus glutarate CAA57200.1 fermentans atoA, atoD P76459.1,Escherichia butanoate P76458.1 coli 13 3.1.2.a 4-hydroxy- 4-hydroxy-4-hydroxybutyryl- tesB NP_414986 Escherichia adipyl-CoA butyratebutyryl-coA CoA hydrolase coli acot12 NP_570103.1 Rattus butyryl-CoAnorvegicus hibch Q6NVY1.2 Homo sapiens 3-hydroxy- propanoyl-CoA 136.2.1.a 4-hydroxy- 4-hydroxy- 4-hydroxy- sucCD NP_415256.1, Escherichiasuccinate butyrate butyryl-coA butyryl-CoA AAC73823.1 coli ligase (or 4-hydroxybutyryl- CoA synthetase) phl CAJ15517.1 Penicillium phenylacetatechrysogenum bioW NP_390902.2 Bacillus 6-carboxy- subtilis hexanoate 131.3.1.a 4-hydroxybut- 4-hydroxy- 4-hydroxybut-2- bcd, etfA, NP_349317.1,Clostridium 2-enoyl-CoA butyryl-CoA enoyl-CoA etfB NP_349315.1,acetobutylicum reductase NP_349316.1 TER Q5EU90.1 Euglena gracilisTDE0597 NP_971211.1 Treponema denticola 8 1.1.1.c 4-hydroxy- 1,4-4-hydroxybutyryl- adhE2 AAK09379.1 Clostridium butanoyl-CoA butyryl-CoAbutanediol CoA reductase acetobutylicum (alcohol forming) mcr AAS20429.1Chloroflexus malonyl-CoA aurantiacus FAR AAD38039.1 Simmondsia longchain chinensis acyl-CoA 8 1.2.1.b 4-hydroxy- 4-hydroxy- 4-hydroxy- sucDP38947.1 Clostridium Succinyl-CoA butyryl-CoA butanal butyryl-CoAkluyveri reductase (or 4- hydroxybutanal dehydrogenase) sucD NP_904963.1Porphyromonas Succinyl-CoA gingivalis Msed_0709 YP_001190808.1Metallosphaera Malonyl-CoA sedula 8 1.1.1.a 4-hydroxy- 1,4-1,4-butanediol ADH2 NP_014032.1 Saccharymyces general butanal butanedioldehydrogenase cerevisiae yqhD NP_417484.1 Escherichia >C3 coli 4hbdL21902.1 Clostridium Succinate kluyveri DSM 555 semialdehyde

EXAMPLE XII BDO Producing Strains Expressing Succinyl-CoA Synthetase

This example describes increased production of BDO in BDO producingstrains expressing succinyl-CoA synthetase.

As discussed above, succinate can be a precursor for production of BDOby conversion to succinyl-CoA (see also WO2008/115840, WO 2009/023493,U.S. publication 2009/0047719, U.S. publication 2009/0075351).Therefore, the host strain was genetically modified to overexpress theE. coli sucCD genes, which encode succinyl-CoA synthetase. Thenucleotide sequence of the E. coli sucCD operon is shown in FIG. 14A,and the amino acid sequences for the encoded succinyl-CoA synthetasesubunits are shown in FIGS. 14B and 14C. Briefly, the E. coli sucCDgenes were cloned by PCR from E. coli chromosomal DNA and introducedinto multicopy plasmids pZS*13, pZA13, and pZE33 behind the PA1lacO-1promoter (Lutz and Bujard, Nucleic Acids Res. 25:1203-1210 (1997)) usingstandard molecular biology procedures.

The E. coli sucCD genes, which encode the succinyl-CoA synthetase, wereoverexpressed. The results showed that introducing into the strainssucCD to express succinyl-CoA synthetase improved BDO production invarious strains compared to either native levels of expression orexpression of cat1, which is a succinyl-CoA/acetyl-CoA transferase.Thus, BDO production was improved by overexpressing the native E. colisucCD genes encoding succinyl-CoA synthetase.

EXAMPLE XIII Expression of Heterologous Genes Encoding BDO PathwayEnzymes

This example describes the expression of various non-native pathwayenzymes to provide improved production of BDO.

Alpha-Ketoglutarate Decarboxylase. The Mycobacterium bovis sucA geneencoding alpha-ketoglutarate decarboxylase was expressed in hoststrains. Overexpression of M. bovis sucA improved BDO production (seealso WO2008/115840, WO 2009/023493, U.S. publication 2009/0047719, U.S.publication 2009/0075351). The nucleotide and amino acid sequences of M.bovis sucA and the encoded alpha-ketoglutarate decarboxylase are shownin FIG. 15.

To construct the M. bovis sucA expressing strains, fragments of the sucAgene encoding the alpha-ketoglutarate decarboxylase were amplified fromthe genomic DNA of Mycobacterium bovis BCG (ATCC 19015; American TypeCulture Collection, Manassas Va.) using primers shown below. Thefull-length gene was assembled by ligation reaction of the fouramplified DNA fragments, and cloned into expression vectors pZS*13 andpZE23 behind the P_(A1lacO-1) promoter (Lutz and Bujard, Nucleic AcidsRes. 25:1203-1210 (1997)). The nucleotide sequence of the assembled genewas verified by DNA sequencing.

Primers for fragment 1: (SEQ ID NO: 3) 5′-ATGTACCGCAAGTTCCGC-3′(SEQ ID NO: 4) 5′-CAATTTGCCGATGCCCAG-3′ Primers for fragment 2:(SEQ ID NO: 5) 5′-GCTGACCACTGAAGACTTTG-3′ (SEQ ID NO: 6)5′-GATCAGGGCTTCGGTGTAG-3′ Primers for fragment 3: (SEQ ID NO: 7)5′-TTGGTGCGGGCCAAGCAGGATCTGCTC-3′ (SEQ ID NO: 8)5′-TCAGCCGAACGCCTCGTCGAGGATCTCCTG-3′ Primers for fragment 4:(SEQ ID NO: 9) 5′-TGGCCAACATAAGTTCACCATTCGGGCAAAAC-3′ (SEQ ID NO: 10)5′-TCTCTTCAACCAGCCATTCGTTTTGCCCG-3′

Functional expression of the alpha-ketoglutarate decarboxylase wasdemonstrated using both in vitro and in vivo assays. The SucA enzymeactivity was measured by following a previously reported method (Tian etal., Proc. Natl. Acad. Sci. USA 102:10670-10675 (2005)). The reactionmixture contained 50 mM potassium phosphate buffer, pH 7.0, 0.2 mMthiamine pyrophosphate, 1 mM MgCl₂, 0.8 mM ferricyanide, 1 mMalpha-ketoglutarate and cell crude lysate. The enzyme activity wasmonitored by the reduction of ferricyanide at 430 nm. The in vivofunction of the SucA enzyme was verified using E. coli whole-cellculture. Single colonies of E. coli MG1655 lacI^(q) transformed withplasmids encoding the SucA enzyme and the 4-hydroxybutyratedehydrogenase (4Hbd) was inoculated into 5 mL of LB medium containingappropriate antibiotics. The cells were cultured at 37° C. overnightaerobically. A 200 uL of this overnight culture was introduced into 8 mLof M9 minimal medium (6.78 g/L Na₂HPO₄, 3.0 g/L KH₂PO₄, 0.5 g/L NaCl,1.0 g/L NH₄Cl, 1 mM MgSO₄, 0.1 mM CaCl₂) supplemented with 20 g/Lglucose, 100 mM 3-(N-morpholino)propanesulfonic acid (MOPS) to improvethe buffering capacity, 10 μg/mL thiamine, and the appropriateantibiotics. Microaerobic conditions were established by initiallyflushing capped anaerobic bottles with nitrogen for 5 minutes, thenpiercing the septum with a 23G needle following inoculation. The needlewas kept in the bottle during growth to allow a small amount of air toenter the bottles. The protein expression was induced with 0.2 mMisopropyl β-D-1-thiogalactopyranoside (IPTG) when the culture reachedmid-log growth phase. As controls, E. coli MG1655 lacI^(q) strainstransformed with only the plasmid encoding the 4-hydroxybutyratedehydrogenase and only the empty vectors were cultured under the samecondition (see Table 23). The accumulation of 4-hydroxybutyrate (4HB) inthe culture medium was monitored using LCMS method. Only the E. colistrain expressing the Mycobacterium alpha-ketoglutarate decarboxylaseproduced significant amount of 4HB (see FIG. 16).

TABLE 24 Three strains containing various plasmid controls and encodingsucA and 4-hydroxybutyrate dehydrogenase. Host pZE13 pZA33 1 MG1655laclq vector vector 2 MG1655 laclq vector 4hbd 3 MG1655 laclq sucA 4hbd

A separate experiment demonstrated that the alpha-ketoglutaratedecarboxylase pathway functions independently of the reductive TCAcycle. E. coli strain ECKh-401 (ΔadhE ΔldhA ΔpflB ΔlpdA::K.p.lpdA322Δmdh ΔarcA) was used as the host strain (see Table 25). All the threeconstructs contained the gene encoding 4HB dehydrogenase (4Hbd).Construct 1 also contained the gene encoding the alpha-ketoglutaratedecarboxylase (sucA). Construct 2 contained the genes encoding thesuccinyl-CoA synthetase (sucCD) and the CoA-dependent succinatesemialdehyde dehydrogenase (sucD), which are required for the synthesisof 4HB via the reductive TCA cycle. Construct 3 contains all the genesfrom 1 and 2. The three E. coli strains were cultured under the sameconditions as described above except the second culture was under themicro-aerobic condition. By expressing the SucA enzyme, construct 3produced more 4HB than construct 2, which relies on the reductive TCAcycle for 4HB synthesis (see FIG. 17).

Further support for the contribution of alpha-ketoglutaratedecarboxylase to production of 4HB and BDO was provided by flux analysisexperiments. Cultures of ECKh-432, which contains both sucCD-sucD andsucA on the chromosome, were grown in M9 minimal medium containing amixture of 1-13C-glucose (60%) and U-13C-glucose (40%). The biomass washarvested, the protein isolated and hydrolyzed to amino acids, and thelabel distribution of the amino acids analyzed by gaschromatography-mass spectrometry (GCMS) as described previously (Fischerand Sauer, Eur. J. Biochem. 270:880-891 (2003)). In addition, the labeldistribution of the secreted 4HB and BDO was analyzed by GCMS asdescribed in WO2008115840 A2. This data was used to calculate theintracellular flux distribution using established methods (Suthers etal., Metab. Eng. 9:387-405 (2007)). The results indicated that between56% and 84% of the alpha-ketoglutarate was channeled throughalpha-ketoglutarate decarboxylase into the BDO pathway. The remainderwas oxidized by alpha-ketoglutarate dehydrogenase, which then enteredBDO via the succinyl-CoA route.

These results demonstrate 4-hydroxybutyrate producing strains thatcontain the sucA gene from Mycobacterium bovis BCG expressed on aplasmid. When the plasmid encoding this gene is not present,4-hydroxybutyrate production is negligible when sucD (CoA-dependantsuccinate semialdehyde dehydrogenase) is not expressed. The M. bovisgene is a close homolog of the Mycobacterium tuberculosis gene whoseenzyme product has been previously characterized (Tian et al., supra,2005).

Succinate semialdehyde dehydrogenase (CoA-dependent), 4-hydroxybutyratedehydrogenase, and 4-hydroxybutyryl-CoA/acetyl-CoA transferase. Thegenes from Porphyromonas gingivalis W83 can be effective components ofthe pathway for 1,4-butanediol production (see also WO2008/115840, WO2009/023493, U.S. publication 2009/0047719, U.S. publication2009/0075351). The nucleotide sequence of CoA-dependent succinatesemialdehyde dehydrogenase (sucD) from Porphyromonas gingivalis is shownin FIG. 18A, and the encoded amino acid sequence is shown in FIG. 18B.The nucleotide sequence of 4-hydroxybutyrate dehydrogenase (4hbd) fromPorphymonas gingivalis is shown in FIG. 19A, and the encoded amino acidsequence is shown in FIG. 19B. The nucleotide sequence of4-hydroxybutyrate CoA transferase (cat2) from Porphyromonas gingivalisis shown in FIG. 20A, and the encoded amino acid sequence is shown inFIG. 20B.

Briefly, the genes from Porphyromonas gingivalis W83 encoding succinatesemialdehyde dehydrogenase (CoA-dependent) and 4-hydroxybutyratedehydrogenase, and in some cases additionally4-hydroxybutyryl-CoA/acetyl-CoA, were cloned by PCR from P. gingivalischromosomal DNA and introduced into multicopy plasmids pZS*13, pZA13,and pZE33 behind the PA1lacO-1 promoter (Lutz and Bujard, Nucleic AcidsRes. 25:1203-1210 (1997)) using standard molecular biology procedures.These plasmids were then introduced into host strains.

The Porphyromonas gingivalis W83 genes were introduced into productionstrains as described above. Some strains included only succinatesemialdehyde dehydrogenase (CoA-dependant) and 4-hydroxybutyratedehydrogenase without 4-hydroxybutyryl-CoA/acetyl-CoA transferase.

Butyrate Kinase and Phosphotransbutyrylase. Butyrate kinase (BK) andphosphotransbutyrylase (PTB) enzymes can be utilized to produce4-hydroxybutyryl-CoA (see also WO2008/115840, WO 2009/023493, U.S.publication 2009/0047719, U.S. publication 2009/0075351). In particular,the Clostridium acetobutylicum genes, buk1 and ptb, can be utilized aspart of a functional BDO pathway.

Initial experiments involved the cloning and expression of the native C.acetobutylicum PTB (020) and BK (021) genes in E. coli. Where required,the start codon and stop codon for each gene were modified to “ATG” and“TAA,” respectively, for more optimal expression in E. coli. The C.acetobutylicum gene sequences (020N and 021N) and their correspondingtranslated peptide sequences are shown in FIGS. 21 and 22.

The PTB and BK genes exist in C. acetobutylicum as an operon, with thePTB (020) gene expressed first. The two genes are connected by thesequence “atta aagttaagtg gaggaatgtt aac” (SEQ ID NO:11) that includes are-initiation ribosomal binding site for the downstream BK (021) gene.The two genes in this context were fused to lac-controlled promoters inexpression vectors for expression in E. coli (Lutz and Bujard, NucleicAcids Res. 25:1203-1210 (1997)).

Expression of the two proteins from these vector constructs was found tobe low in comparison with other exogenously expressed genes due to thehigh incidence of codons in the C. acetobutylicum genes that occur onlyrarely in E. coli. Therefore new 020 and 021 genes were predicted thatchanged rare codons for alternates that are more highly represented inE. coli gene sequences. This method of codon optimization followedalgorithms described previously (Sivaraman et al., Nucleic Acids Res.36:e16 (2008)). This method predicts codon replacements in context withtheir frequency of occurrence when flanked by certain codons on eitherside. Alternative gene sequences for 020 (FIG. 23) and 021 (FIG. 24)were determined in which increasing numbers of rare codons were replacedby more prevalent codons (A<B<C<D) based on their incidence in theneighboring codon context. No changes in actual peptide sequencecompared to the native 020 and 021 peptide sequences were introduced inthese predicted sequences.

The improvement in expression of the BK and PTB proteins resulting fromcodon optimization is shown in FIG. 25A. Expression of the native genesequences is shown in lane 2, while expression of the 020B-021B and020C-021C is shown in lanes 3 and 4, respectively. Higher levels ofprotein expression in the codon-optimized operons 020B-021B (2021B) and020C-021C (2021 C) also resulted in increased activity compared to thenative operon (2021n) in equivalently-expressed E. coli crude extracts(FIG. 25B).

The codon optimized operons were expressed on a plasmid in strainECKh-432 (ΔadhE ΔldhA ΔpflB ΔlpdA::K.p.lpdA322 Δmdh ΔarcA gltAR163LfimD:: E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd fimD:: M.bovis sucA, C. kluyveri 4hbd) along with the C. acetobutylicum aldehydedehydrogenase to provide a complete BDO pathway. Cells were cultured inM9 minimal medium containing 20 g/L glucose, using a 23G needle tomaintain microaerobic conditions as described above. The resultingconversion of glucose to the final product BDO was measured. Alsomeasured was the accumulation of gamma-butyrylactone (GBL), which is aspontaneously rearranged molecule derived from 4Hb-CoA, the immediateproduct of the PTB-BK enzyme pair. FIG. 26 shows that expression of thenative 2021n operon resulted in comparable BDO levels to an alternativeenzyme function, Cat2 (034), that is capable of converting 4HB and freeCoA to 4HB-CoA. GBL levels of 034 were significantly higher than 2021n,suggesting that the former enzyme has more activity than PTB-BKexpressed from the native genes. However levels of both BDO and GBL werehigher than either 034 or 2021n when the codon-optimized variants 2021Band 2021C were expressed, indicating that codon optimization of thegenes for PTB and BK significantly increases their contributions to BDOsynthesis in E. coli.

These results demonstrate that butyrate kinase (BK) andphosphotransbutyrylase (PTB) enzymes can be employed to convert4-hydroxybutyrate to 4-hydroxybutyryl-CoA. This eliminates the need fora transferase enzyme such as 4-hydroxybutyryl-CoA/Acetyl-CoAtransferase, which would generate one mole of acetate per mol of4-hydroxybutyryl-CoA produced. The enzymes from Clostridiumacetobutylicum are present in a number of engineered strains for BDOproduction.

4-hydroxybutyryl-CoA Reductase. The Clostridium beijerinckii ald genecan be utilized as part of a functional BDO pathway (see alsoWO2008/115840, WO 2009/023493, U.S. publication 2009/0047719, U.S.publication 2009/0075351). The Clostridium beijerinckii ald can also beutilized to lower ethanol production in BDO producing strains.Additionally, a specific codon-optimized ald variant (GNM0025B) wasfound to improve BDO production.

The native C. beijerinckii ald gene (025n) and the predicted proteinsequence of the enzyme are shown in FIG. 27. As was seen for theClostridium acetobutylicum PTB and BK genes, expression of the native C.beijerinckii ald gene was very low in E. coli. Therefore, fourcodon-optimized variants for this gene were predicted. FIGS. 28A-28Dshow alternative gene sequences for 025, in which increasing numbers ofrare codons are replaced by more prevalent codons (A<B<C<D) based ontheir incidence in the neighboring codon context (25A, P=0.05; 25B,P=0.1; 25C, P=0.15; 25D, P=1). No changes in actual peptide sequencecompared to the native 025 peptide sequence were introduced in thesepredictions. Codon optimization significantly increased expression ofthe C. beijerinckii ald (see FIG. 29), which resulted in significantlyhigher conversion of glucose to BDO in cells expressing the entire BDOpathway (FIG. 30A).

The native and codon-optimized genes were expressed on a plasmid alongwith P. gingivalis Cat2, in the host strain ECKh-432 (ΔadhE ΔldhA ΔpflBΔlpdA::K.p.lpdA322 Δmdh ΔarcA gltAR163L ΔackA fimD:: E. coli sucCD, P.gingivalis sucD, P. gingivalis 4hbd fimD:: M. bovis sucA, C. kluyveri4hbd), thus containing a complete BDO pathway. Cells were culturedmicroaerobically in M9 minimal medium containing 20 g/L glucose asdescribed above. The relative production of BDO and ethanol by the C.beijerinckii Ald enzyme (expressed from codon-optimized variant gene025B) was compared with the C. acetobutylicum AdhE2 enzyme (see FIG.30B). The C. acetobutylicum AdhE2 enzyme (002C) produced nearly 4 timesmore ethanol than BDO. In comparison, the C. beijerinckii Ald (025B) (inconjunction with an endogenous ADH activity) produced equivalent amountsof BDO, yet the ratio of BDO to ethanol production was reversed for thisenzyme compared to 002C. This suggests that the C. beijerinckii Ald ismore specific for 4HB-CoA over acetyl-coA than the C. acetobutylicumAdhE2, and therefore the former is the preferred enzyme for inclusion inthe BDO pathway.

The Clostridium beijerinckii ald gene (Toth et al., Appl. Environ.Microbiol. 65:4973-4980 (1999)) was tested as a candidate for catalyzingthe conversion of 4-hydroxybutyryl-CoA to 4-hydroxybutanal. Over fiftyaldehyde dehydrogenases were screened for their ability to catalyze theconversion of 4-hydroxybutyryl-CoA to 4-hydroxybutyraldehyde. The C.beijerinckii ald gene was chosen for implementation into BDO-producingstrains due to the preference of this enzyme for 4-hydroxybutyryl-CoA asa substrate as opposed to acetyl-CoA. This is important because mostother enzymes with aldehyde dehydrogenase functionality (for example,adhE2 from C. acetobutylicum (Fontaine et al., J Bacteriol. 184:821-830(2002)) preferentially convert acetyl-CoA to acetaldehyde, which in turnis converted to ethanol. Utilization of the C. beijerinckii gene lowersthe amount of ethanol produced as a byproduct in BDO-producingorganisms. Also, a codon-optimized version of this gene expresses verywell in E. coli (Sivaraman et al., Nucleic Acids Res. 36:e16 (2008)).

4-hydroxybutanal Reductase. 4-hydroxybutanal reductase activity of adh1from Geobacillus thermoglucosidasius (M10EXG) was utilized. This led toimproved BDO production by increasing 4-hydroxybutanal reductaseactivity over endogenous levels.

Multiple alcohol dehydrogenases were screened for their ability tocatalyze the reduction of 4-hydroxybutanal to BDO. Most alcoholdehydrogenases with high activity on butyraldehyde exhibited far loweractivity on 4-hydroxybutyraldehyde. One notable exception is the adh1gene from Geobacillus thermoglucosidasius M10EXG (Jeon et al., J.Biotechnol. 135:127-133 (2008)) (GNM0084), which exhibits high activityon both 4-hydroxybutanal and butanal.

The native gene sequence and encoded protein sequence if the adh1 genefrom Geobacillus thermoglucosidasius are shown in FIG. 31. The G.thermoglucosidasius ald1 gene was expressed in E. coli.

The Adh1 enzyme (084) expressed very well from its native gene in E.coli (see FIG. 32A). In ADH enzyme assays, the E. coli expressed enzymeshowed very high reductive activity when butyraldehyde or 4HB-aldehydewere used as the substrates (see FIG. 32B). The Km values determined forthese substrates were 1.2 mM and 4.0 mM, respectively. These activityvalues showed that the Adh1 enzyme was the most active on reduction of4HB-aldehyde of all the candidates tested.

The 084 enzyme was tested for its ability to boost BDO production whencoupled with the C. beijerinckii ald. The 084 gene was inserted behindthe C. beijerinckii ald variant 025B gene to create a synthetic operonthat results in coupled expression of both genes. Similar constructslinked 025B with other ADH candidate genes, and the effect of includingeach ADH with 025B on BDO production was tested. The host strain usedwas ECKh-459 (ΔadhE ldhA ΔpflB ΔlpdA::fnr-pflB6-K.p.lpdA322 Δmdh ΔarcAgltAR163L fimD:: E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbdfimD:: M. bovis sucA, C. kluyveri 4hbd fimD:: C. acetobutylicum buk1, C.acetobutylicum ptb), which contains the remainder of the BDO pathway onthe chromosome. The 084 ADH expressed in conjunction with 025B showedthe highest amount of BDO (right arrow in FIG. 33) when compared with025B only (left arrow in FIG. 33) and in conjunction with endogenous ADHfunctions. It also produced more BDO than did other ADH enzymes whenpaired with 025B, indicated as follows: 026A-C, codon-optimized variantsof Clostridium acetobutylicum butanol dehydrogenase; 050, Zymomonasmobilis alcohol dehydrogenase I; 052, Citrobacter freundii1,3-propanediol dehydrogenase; 053, Lactobacillus brevis 1,3-propanedioldehydrogenase; 057, Bacteroides fragilis lactaldehyde reductase; 058, E.coli 1,3-propanediol dehydrogenase; 071, Bacillus subtilis 168alpha-ketoglutarate semialdehyde dehydrogenase. The constructs labeled“PT5lacO” are those in which the genes are driven by the PT5lacOpromoter. In all other cases, the PA1lacO-1 promoter was used. Thisshows that inclusion of the 084 ADH in the BDO pathway increased BDOproduction.

EXAMPLE XIV BDO Producing Strains Expressing Pyruvate Dehydrogenase

This example describes the utilization of pyruvate dehydrogenase (PDH)to enhance BDO production. Heterologous expression of the Klebsiellapneumonia lpdA gene was used to enhance BDO production.

Computationally, the NADH-generating conversion of pyruvate toacetyl-CoA is required to reach the maximum theoretical yield of1,4-butanediol (see also WO2008/115840, WO 2009/023493, U.S. publication2009/0047719, U.S. publication 2009/0075351; WO 2008/018930; Kim et al.,Appl. Environ. Microbiol. 73:1766-1771 (2007); Kim et al., J. Bacteriol.190:3851-3858 (2008); Menzel et al., J. Biotechnol. 56:135-142 (1997)).Lack of PDH activity was shown to reduce the maximum anaerobictheoretical yield of BDO by 11% if phosphoenolpyruvate carboxykinase(PEPCK) activity cannot be attained and by 3% if PEPCK activity can beattained. More importantly, however, absence of PDH activity in theOptKnock strain #439, described in WO 2009/023493 and U.S. publication2009/0047719, which has the knockout of ADHEr, ASPT, LDH_D, MDH andPFLi, would reduce the maximum anaerobic yield of BDO by 54% or by 43%if PEPCK activity is absent or present, respectively. In the presence ofan external electron acceptor, lack of PDH activity would reduce themaximum yield of the knockout strain by 10% or by 3% assuming that PEPCKactivity is absent or present, respectively.

PDH is one of the most complicated enzymes of central metabolism and iscomprised of 24 copies of pyruvate decarboxylase (E1) and 12 moleculesof dihydrolipoyl dehydrogenase (E3), which bind to the outside of thedihydrolipoyl transacetylase (E2) core. PDH is inhibited by highNADH/NAD, ATP/ADP, and Acetyl-CoA/CoA ratios. The enzyme naturallyexhibits very low activity under oxygen-limited or anaerobic conditionsin organisms such as E. coli due in large part to the NADH sensitivityof E3, encoded by lpdA. To this end, an NADH-insensitive version of thelpdA gene from Klebsiella pneumonia was cloned and expressed to increasethe activity of PDH under conditions where the NADH/NAD ratio isexpected to be high.

Replacement of the Native lpdA. The pyruvate dehydrogenase operon ofKlebsiella pneumoniae is between 78 and 95% identical at the nucleotidelevel to the equivalent operon of E. coli. It was shown previously thatK. pneumoniae has the ability to grow anaerobically in presence ofglycerol (Menzel et al., J. Biotechnol. 56:135-142 (1997); Menzel etal., Biotechnol. Bioeng. 60:617-626 (1998)). It has also been shown thattwo mutations in the lpdA gene of the operon of E. coli would increaseits ability to grow anaerobically (Kim et al. Appl. Environ. Microbiol.73:1766-1771 (2007); Kim et al., J. Bacteriol. 190:3851-3858 (2008)).The lpdA gene of K. pneumonia was amplified by PCR using genomic DNA(ATCC700721D) as template and the primers KP-lpdA-Bam(5′-acacgcggatccaacgtcccgg-3′) (SEQ ID NO:12) and KP-lpdA-Nhe(5′-agcggctccgctagccgcttatg-3′) (SEQ ID NO:13). The resulting fragmentwas cloned into the vector pCR-BluntII-TOPO (Invitrogen; CarlsbadCalif.), leading to plasmid pCR-KP-lpdA.

The chromosomal gene replacement was performed using a non-replicativeplasmid and the sacB gene from Bacillus subtilis as a means ofcounterselection (Gay et al., J. Bacteriol. 153:1424-1431 (1983)). Thevector used is pRE118 (ATCC87693) deleted of the oriT and IS sequences,which is 3.6 kb in size and carrying the kanamycin resistance gene. Thesequence was confirmed, and the vector was called pRE118-V2 (see FIG.34).

The E. coli fragments flanking the lpdA gene were amplified by PCR usingthe combination of primers: EC-aceF-Pst (5′-aagccgttgctgcagctcttgagc-3′)(SEQ ID NO:14)+EC-aceF-Bam2 (5′-atctccggcggtcggatccgtcg-3′) (SEQ IDNO:15) and EC-yacH-Nhe (5′-aaagcggctagccacgccgc-3′) (SEQ IDNO:16)+EC-yacH-Kpn (5′-attacacgaggtacccaacg-3′) (SEQ ID NO:17). ABamHI-XbaI fragment containing the lpdA gene of K. pneumonia wasisolated from plasmid pCR-KP-lpdA and was then ligated to the above E.coli fragments digested with PstI+BamHI and NheI-KpnI respectively, andthe pRE118-V2 plasmid digested with KpnI and PstI. The resulting plasmid(called pRE118-M2.1 lpdA yac) was subjected to Site Directed Mutagenesis(SDM) using the combination of primers KP-lpdA-HisTyr-F(5′-atgctggcgtacaaaggtgtcc-3′) (SEQ ID NO:18) and(5′-ggacacctttgtacgccagcat-3′) (SEQ ID NO:19) for the mutation of theHis 322 residue to a Tyr residue or primers KP-lpdA-GluLys-F(5′-atcgcctacactaaaccagaagtgg-3′) (SEQ ID NO:20) and KP-lpdA-GluLys-R(5′-ccacttctggtttagtgtaggcgat-3′) (SEQ ID NO:21) for the mutation of theresidue Glu 354 to Lys residue. PCR was performed with the PolymerasePfu Turbo (Stratagene; San Diego Calif.). The sequence of the entirefragment as well as the presence of only the desired mutations wasverified. The resulting plasmid was introduced into electro competentcells of E. coli ΔadhE::Frt-ΔldhA::Frt by transformation. The firstintegration event in the chromosome was selected on LB agar platescontaining Kanamycin (25 or 50 mg/L). Correct insertions were verifiedby PCR using 2 primers, one located outside the region of insertion andone in the kanamycin gene (5′-aggcagttccataggatggc-3′) (SEQ ID NO:22).Clones with the correct insertion were selected for resolution. Theywere sub-cultured twice in plain liquid LB at the desired temperatureand serial dilutions were plated on LB-no salt-sucrose 10% plates.Clones that grew on sucrose containing plates were screened for the lossof the kanamycin resistance gene on LB-low salt agar medium and the lpdAgene replacement was verified by PCR and sequencing of the encompassingregion. Sequence of the insertion region was verified, and is asdescribed below. One clone (named 4-4-P1) with mutation Glu354Lys wasselected. This clone was then transduced with P1 lysate of E. coliΔPflB::Frt leading to strain ECKh-138 (ΔadhE ΔldhA ΔpflBΔlpdA::K.p.lpdA322).

The sequence of the ECKh-138 region encompassing the aceF and lpdA genesis shown in FIG. 35. The K. pneumonia lpdA gene is underlined, and thecodon changed in the Glu354Lys mutant shaded. The protein sequencecomparison of the native E. coli lpdA and the mutant K. pneumonia lpdAis shown in FIG. 36.

To evaluate the benefit of using K. pneumoniae lpdA in a BDO productionstrain, the host strains AB3 and ECKh-138 were transformed with plasmidsexpressing the entire BDO pathway from strong, inducible promoters.Specifically, E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd wereexpressed on the medium copy plasmid pZA33, and P. gingivalis Cat2 andC. acetobutylicum AdhE2 were expressed on the high copy plasmid pZE13.These plasmids have been described in the literature (Lutz and H.Bujard, Nucleic Acids Res 25:1203-1210 (1997)), and their use for BDOpathway expression is described in Example XIII and WO2008/115840.

Cells were grown anaerobically at 37° C. in M9 minimal medium (6.78 g/LNa₂HPO₄, 3.0 g/L KH₂PO₄, 0.5 g/L NaCl, 1.0 g/L NH₄Cl, 1 mM MgSO₄, 0.1 mMCaCl₂) supplemented with 20 g/L glucose, 100 mM3-(N-morpholino)propanesulfonic acid (MOPS) to improve the bufferingcapacity, 10 μg/mL thiamine, and the appropriate antibiotics.Microaerobic conditions were established by initially flushing cappedanaerobic bottles with nitrogen for 5 minutes, then piercing the septumwith a 23G needle following inoculation. The needle was kept in thebottle during growth to allow a small amount of air to enter thebottles. 0.25 mM IPTG was added when OD600 reached approximately 0.2 toinduce the pathway genes, and samples taken for analysis every 24 hoursfollowing induction. The culture supernatants were analyzed for BDO,4HB, and other by-products as described in Example II and inWO2008/115840. BDO and 4HB production in ECKh-138 was significantlyhigher after 48 hours than in AB3 or the host used in previous work,MG1655 ΔldhA (FIG. 37).

PDH promoter replacement. It was previously shown that the replacementof the pdhR repressor by a transcriptional fusion containing the Fnrbinding site, one of the pflB promoters, and its ribosome binding site(RBS), thus leading to expression of the aceEF-lpd operon by ananaerobic promoter, should increase pdh activity anaerobically (Zhou etal., Biotechnol. Lett. 30:335-342 (2008)). A fusion containing the Fnrbinding site, the pflB-p6 promoter and an RBS binding site wereconstructed by overlapping PCR. Two fragments were amplified, one usingthe primers aceE-upstream-RC(5′-tgacatgtaacacctaccttctgtgcctgtgccagtggttgctgtgatatagaag-3′) (SEQ IDNO:23) and pflBp6-Up-Nde(5′-ataataatacatatgaaccatgcgagttacgggcctataagccaggcg-3′) (SEQ ID NO:24)and the other using primers aceE-EcoRV-EC(5′-agtttttcgatatctgcatcagacaccggcacattgaaacgg-3′) (SEQ ID NO:25) andaceE-upstream(5′-ctggcacaggcacagaaggtaggtgttacatgtcagaacgtttacacaatgacgtggatc-3′)(SEQ ID NO:26). The tw fragments were assembled by overlapping PCR, andthe final DNA fragment was digested with the restriction enzymes NdeIand BamHI. This fragment was subsequently introduced upstream of theaceE gene of the E. coli operon using pRE118-V2 as described above. Thereplacement was done in strains ECKh-138 and ECKh-422. The nucleotidesequence encompassing the 5′ region of the aceE gene was verified and isshown in FIG. 37. FIG. 37 shows the nucleotide sequence of 5′ end of theaceE gene fused to the pflB-p6 promoter and ribosome binding site (RBS).The 5′ italicized sequence shows the start of the aroP gene, which istranscribed in the opposite direction from the pdh operon. The 3′italicized sequence shows the start of the aceE gene. In upper case:pflB RBS. Underlined: FNR binding site. In bold: pflB-p6 promotersequence.

lpdA Promoter Replacement. The promoter region containing the fnrbinding site, the pflB-p6 promoter and the RBS of the pflB gene wasamplified by PCR using chromosomal DNA template and primersaceF-pflBp6-fwd(5′-agacaaatcggttgccgtttgttaagccaggcgagatatgatctatatc-3′) (SEQ ID NO:27)and lpdA-RBS-B-rev(5′-gagttttgatttcagtactcatcatgtaacacctaccttettgctgtgatatag-3′) (SEQ IDNO:28). Plasmid 2-4-a was amplified by PCR using primers B-RBS-lpdA fwd(5′-ctatatcacagcaagaaggtaggtgttacatgatgagtactgaaatcaaaactc-3′) (SEQ IDNO:29) and pflBp6-aceF-rev(5′-gatatagatcatatctcgcctggcttaacaaacggcaaccgatttgtct-3′) (SEQ IDNO:30). The two resulting fragments were assembled using the BPS cloningkit (BPS Bioscience; San Diego Calif.). The resulting construct wassequenced verified and introduced into strain ECKh-439 using thepRE118-V2 method described above. The nucleotide sequence encompassingthe aceF-lpdA region in the resulting strain ECKh-456 is shown in FIG.39.

The host strain ECKh-439 (ΔadhE ΔldhA ΔpflB ΔlpdA::K.p.lpdA322 ΔmdhΔarcA gltAR163L ackA fimD:: E. coli sucCD, P. gingivalis sucD, P.gingivalis 4hbd fimD:: M. bovis sucA, C. kluyveri 4hbd), theconstruction of which is described below, and the pdhR and lpdA promoterreplacement derivatives ECKh-455 and ECKh-456, were tested for BDOproduction. The strains were transformed with pZS*13 containing P.gingivalis Cat2 and C. beijerinckii Ald to provide a complete BDOpathway. Cells were cultured in M9 minimal medium supplemented with 20g/L glucose as described above. 48 hours after induction with 0.2 mMIPTG, the concentrations of BDO, 4HB, and pyruvate were as shown in FIG.40. The promoter replacement strains produce slightly more BDO than theisogenic parent.

These results demonstrated that expression of pyruvate dehydrogenaseincreased production of BDO in BDO producing strains.

EXAMPLE XV BDO Producing Strains Expressing Citrate Synthase andAconitase

This example describes increasing activity of citrate synthase andaconitase to increase production of BDO. An R163L mutation into gltA wasfound to improve BDO production. Additionally, an arcA knockout was usedto improve BDO production.

Computationally, it was determined that flux through citrate synthase(CS) and aconitase (ACONT) is required to reach the maximum theoreticalyield of 1,4-butanediol (see also WO2008/115840, WO 2009/023493, U.S.publication 2009/0047719, U.S. publication 2009/0075351). Lack of CS orACONT activity would reduce the maximum theoretical yield by 14% underanaerobic conditions. In the presence of an external electron acceptor,the maximum yield is reduced by 9% or by 6% without flux through CS orACONT assuming the absence or presence of PEPCK activity, respectively.As with pyruvate dehydrogenase (PDH), the importance of CS and ACONT isgreatly amplified in the knockout strain background in which ADHEr,ASPT, LDH_D, MDH and PFLi are knocked out (design #439) (see WO2009/023493 and U.S. publication 2009/0047719, which is incorporatedherein by reference).

The minimal OptKnock strain design described in WO 2009/023493 and U.S.publication 2009/0047719 had one additional deletion beyond ECKh-138,the mdh gene, encoding malate dehydrogenase. Deletion of this gene isintended to prevent flux to succinate via the reductive TCA cycle. Themdh deletion was performed using the λ red homologeous recombinationmethod (Datsenko and Wanner, Proc. Natl. Acad. Sci. USA 97:6640-6645(2000)). The following oligonucleotides were used to PCR amplify thechloramphenicol resistance gene (CAT) flanked by FRT sites from pKD3:

S-mdh-Kan (SEQ ID NO: 31) 5′- TAT TGT GCA TAC AGA TGA ATT TTT ATG CAA ACA GTC AGC CCTGAA GAA GGG TGT AGG CTG GAG CTG CTT C - 3′ AS-mdh-Kan (SEQ ID NO: 32) 5′- CAA AAA ACC GGA GTC TGT GCT CCG GTT TTT TAT TAT CCG CTAATC AAT TAC ATA TGA ATA TCC TCC TTA G - 3′.

Underlined regions indicate homology to pKD3 plasmid and bold sequencerefers to sequence homology upstream and downstream of the mdh ORF.After purification, the PCR product was electroporated into ECKh-138electrocompetent cells that had been transformed with pRedET (tet) andprepared according to the manufacturer's instructions(www.genebridges.com/gb/pdf/K001%20Q %20E %20BAC %20Modification%20Kit-version2.6-2007-screen.pdf). The PCR product was designed so thatit integrated into the ECKh-138 genome at a region upstream of the mdhgene, as shown in FIG. 41.

Recombinants were selected for chloramphenicol resistance and streakpurified. Loss of the mdh gene and insertion of CAT was verified bydiagnostic PCR. To remove the CAT gene, a temperature sensitive plasmidpCP20 containing a FLP recombinase (Datsenko and Wanner, Proc. Natl.Acad. Sci. USA 97:6640-6645 (2000)) was transformed into the cell at 30°C. and selected for ampicillin resistance (AMP). Transformants weregrown nonselectively at 42° C. overnight to thermally induce FLPsynthesis and to cause lose of the plasmid. The culture was then streakpurified, and individual colonies were tested for loss of all antibioticresistances. The majority lost the FRT-flanked resistance gene and theFLP helper plasmid simultaneously. There was also a “FRT” scar leftover.The resulting strain was named ECKh-172.

CS and ACONT are not highly active or highly expressed under anaerobicconditions. To this end, the arcA gene, which encodes for a globalregulator of the TCA cycle, was deleted. ArcA works during microaerobicconditions to induce the expression of gene products that allow theactivity of central metabolism enzymes that are sensitive to low oxygenlevels, aceE, pflB and adhE. It was shown that microaerobically, adeletion in arcA/arcB increases the specific activities of ldh, icd,gltA, mdh, and gdh genes (Salmon et al., J. Biol. Chem. 280:15084-15096(2005); Shalel-Levanon et al., Biotechnol. Bioeng. 92(2):147-159 (2005).The upstream and downstream regions of the arcA gene of E. coli MG1655were amplified by PCR using primers ArcA-up-EcoRI(5′-ataataatagaattcgtttgctacctaaattgccaactaaatcgaaacagg-3′) (SEQ IDNO:33) with ArcA-up-KpnI(5′-tattattatggtaccaatatcatgcagcaaacggtgcaacattgccg-3′) (SEQ ID NO:34)and ArcA-down-EcoRI(5′-tgatctggaagaattcatcggctttaccaccgtcaaaaaaaacggcg-3′) (SEQ ID NO:35)with ArcA-down-PstI(5′-ataaaaccctgcagcggaaacgaagttttatccatttttggttacctg-3′) (SEQ ID NO:36),respectively. These fragments were subsequently digested with therestriction enzymes EcoRI and KpnI (upstream fragment) and EcoRI andPstI (downstream). They were then ligated into the pRE118-V2 plasmiddigested with PstI and KpnI, leading to plasmid pRE118-ΔarcA. Thesequence of plasmid pRE118-ΔarcA was verified. pRE118-ΔarcA wasintroduced into electro-competent cells of E. coli strain ECKh-172(ΔadhE ΔldhA ΔpflB ΔlpdA::K.p.lpdA322 Δmdh). After integration andresolution on LB-no salt-sucrose plates as described above, the deletionof the arcA gene in the chromosome of the resulting strain ECKh-401 wasverified by sequencing and is shown in FIG. 42.

The gltA gene of E. coli encodes for a citrate synthase. It waspreviously shown that this gene is inhibited allosterically by NADH, andthe amino acids involved in this inhibition have been identified(Pereira et al., J. Biol. Chem. 269(1):412-417 (1994); Stokell et al.,J. Biol. Chem. 278(37):35435-35443 (2003)). The gltA gene of E. coliMG1655 was amplified by PCR using primers gltA-up(5′-ggaagagaggctggtacccagaagccacagcagga-3′) (SEQ ID NO:37) and gltA-PstI(5′-gtaatcactgcgtaagcgccatgccccggcgttaattc-3′) (SEQ ID NO:38). Theamplified fragment was cloned into pRE118-V2 after digestion with KpnIand PstI. The resulting plasmid was called pRE118-gltA. This plasmid wasthen subjected to site directed mutagensis (SDM) using primers R163L-f(5′-attgccgcgttcctcctgctgtcga-3′) (SEQ ID NO:39) and R163L-r(5′-cgacagcaggaggaacgcggcaat-3′) (SEQ ID NO:40) to change the residueArg 163 to a Lys residue. The sequence of the entire fragment wasverified by sequencing. A variation of the λ red homologeousrecombination method (Datsenko and Wanner, Proc. Natl. Acad. Sci. USA97:6640-6645 (2000)) was used to replace the native gltA gene with theR163L mutant allele without leaving a Frt scar. The generalrecombination procedure is the same as used to make the mdh deletiondescribed above. First, the strain ECKh-172 was made streptomycinresistant by introducing an rpsL null mutation using the λ redhomologeous recombination method. Next, a recombination was done toreplace the entire wild-type gltA coding region in this strain with acassette comprised of a kanamycin resistance gene (kanR) and a wild-typecopy of the E. coli rpsL gene. When introduced into an E. coli strainharboring an rpsL null mutation, the cassette causes the cells to changefrom resistance to the drug streptomycin to streptomycin sensitivity.DNA fragments were then introduced that included each of the mutantversions of the gltA gene along with appropriate homologous ends, andresulting colony growth was tested in the presence of streptomycin. Thisselected for strains in which the kanR/rpsL cassette had been replacedby the mutant gltA gene. Insertion of the mutant gene in the correctlocus was confirmed by PCR and DNA sequencing analyses. The resultingstrain was called ECKh-422, and has the genotype ΔadhE ΔldhA ΔpflBΔlpdA::K.p.lpdA322 Δmdh ΔarcA gltAR163L. The region encompassing themutated gltA gene of strain ECKh-422 was verified by sequencing, asshown in FIG. 43.

Crude extracts of the strains ECKh-401 and the gltAR163L mutant ECKh-422were then evaluated for citrate synthase activity. Cells were harvestedby centrifugation at 4,500 rpm (Beckman-Coulter, Allegera X-15R;Fullerton Calif.) for 10 min. The pellets were resuspended in 0.3 mLBugBuster (Novagen/EMD; San Diego Calif.) reagent with benzonase andlysozyme, and lysis proceeded for 15 minutes at room temperature withgentle shaking Cell-free lysate was obtained by centrifugation at 14,000rpm (Eppendorf centrifuge 5402; Hamburg Germany) for 30 min at 4° C.Cell protein in the sample was determined using the method of Bradford(Bradford, Anal. Biochem. 72:248-254 (1976)).

Citrate synthase activity was determined by following the formation offree coenzyme A (HS-CoA), which is released from the reaction ofacetyl-CoA with oxaloacetate. The free thiol group of HS-CoA reacts with5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) to form5-thio-2-nitrobenzoic acid (TNB). The concentration of TNB is thenmonitored spectrophotometrically by measuring the absorbance at 410 nm(maximum at 412 nm). The assay mixture contained 100 mM Tris/HCl buffer(pH 7.5), 20 mM acetyl-CoA, 10 mM DTNB, and 20 mM oxaloacetate. For theevaluation of NADH inhibition, 0.4 mM NADH was also added to thereaction. The assay was started by adding 5 microliters of the cellextract, and the rate of reaction was measured by following theabsorbance change over time. A unit of specific activity is defined asthe μmmol of product converted per minute per mg protein.

FIG. 44 shows the citrate synthase activity of wild type gltA geneproduct and the R163L mutant. The assay was performed in the absence orpresence of 0.4 mM NADH.

Strains ECKh-401 and ECKh-422 were transformed with plasmids expressingthe entire BDO pathway. E. coli sucCD, P. gingivalis sucD, P. gingivalis4hbd, and M. bovis sucA were expressed on the low copy plasmid pZS*13,and P. gingivalis Cat2 and C. acetobutylicum AdhE2 were expressed on themedium copy plasmid pZE23. Cultures of these strains were grownmicroaerobically in M9 minimal medium supplemented with 20 g/L glucoseand the appropriate antibiotics as described above. The 4HB and BDOconcentrations at 48 hours post-induction averaged from duplicatecultures are shown in FIG. 45. Both are higher in ECKh-422 than inECKh-401, demonstrating that the enhanced citrate synthase activity dueto the gltA mutation results in increased flux to the BDO pathway.

The host strain modifications described in this section were intended toredirect carbon flux through the oxidative TCA cycle, which isconsistent with the OptKnock strain design described in WO 2009/023493and U.S. publication 2009/0047719. To demonstrate that flux was indeedrouted through this pathway, ¹³C flux analysis was performed using thestrain ECKh-432, which is a version of ECKh-422 in which the upstreampathway is integrated into the chromosome (as described in ExampleXVII). To complete the BDO pathway, P. gingivalis Cat2 and C.beijerinckii Ald were expressed from pZS*13. Four parallel cultures weregrown in M9 minimal medium (6.78 g/L Na₂HPO₄, 3.0 g/L KH₂PO₄, 0.5 g/LNaCl, 1.0 g/L NH₄Cl, 1 mM MgSO₄, 0.1 mM CaCl₂) containing 4 g/L totalglucose of four different labeling ratios (¹⁻¹³C, only the first carbonatom in the glucose molecule is labeled with ¹³C; uniform-¹³C, allcarbon atoms are ¹³C):

1. 80 mol % unlabeled, 20 mol % uniform-¹³C

2. 10 mol % unlabeled, 90 mol % uniform-¹³C

3. 90 mol % ¹⁻¹³C, 10 mol % uniform-¹³C

4. 40 mol % ¹⁻¹³C, 60 mol % uniform-¹³C

Parallel unlabeled cultures were grown in duplicate, from which frequentsamples were taken to evaluate growth rate, glucose uptake rate, andproduct formation rates. In late exponential phase, the labeled cultureswere harvested, the protein isolated and hydrolyzed to amino acids, andthe label distribution of the amino acids analyzed by gaschromatography-mass spectrometry (GCMS) as described previously (Fischerand Sauer, Eur. J. Biochem. 270:880-891 (2003)). In addition, the labeldistribution of the secreted 4HB and BDO in the broth from the labeledcultures was analyzed by GCMS as described in WO2008115840. This datawas collectively used to calculate the intracellular flux distributionusing established methods (Suthers et al., Metab. Eng. 9:387-405(2007)). The resulting central metabolic fluxes and associated 95%confidence intervals are shown in FIG. 46. Values are molar fluxesnormalized to a glucose uptake rate of 1 mmol/hr. The result indicatesthat carbon flux is routed through citrate synthase in the oxidativedirection, and that most of the carbon enters the BDO pathway ratherthan completing the TCA cycle. Furthermore, it confirms there isessentially no flux between malate and oxaloacetate due to the mdhdeletion in this strain.

The advantage of using a knockout strain such as strains designed usingOptKnock for BDO production (see WO 2009/023493 and U.S. publication2009/0047719) can be observed by comparing typical fermentation profilesof ECKh-422 with that of the original strain ECKh-138, in which BDO isproduced from succinate via the reductive TCA cycle (see FIG. 47).Fermentations were performed with 1 L initial culture volume in 2 LBiostat B+ bioreactors (Sartorius; Cedex France) using M9 minimal mediumsupplemented with 20 g/L glucose. The temperature was controlled at 37°C., and the pH was controlled at 7.0 using 2 M NH₄OH or Na₂CO₃. Cellswere grown aerobically to an OD600 of approximately 10, at which timethe cultures were induced with 0.2 mM IPTG. One hour followinginduction, the air flow rate was reduced to 0.02 standard liters perminute for microaerobic conditions. The agitation rate was set at 700rpm. Concentrated glucose was fed to maintain glucose concentration inthe vessel between 0.5 and 10 g/L. Both strains were transformed withplasmids bearing the entire BDO pathway, as in the examples above. InECKh-138, acetate, pyruvate, and 4HB dominate the fermentation, whilewith ECKh-422 BDO is the major product.

EXAMPLE XVI BDO Strains Expression Phosphoenolpyruvate Carboxykinase

This example describes the utilization of phosphoenolpyruvatecarboxykinase (PEPCK) to enhance BDO production. The Haemophilusinfluenza PEPCK gene was used for heterologous expression.

Computationally, it was demonstrated that the ATP-generating conversionof oxaloacetate to phosphoenolpyruvate is required to reach the maximumtheoretical yield of 1,4-butanediol (see also WO2008/115840, WO2009/023493, U.S. publication 2009/0047719, U.S. publication2009/0075351). Lack of PEPCK activity was shown to reduce the maximumtheoretical yield of BDO by 12% assuming anaerobic conditions and by 3%assuming an external electron acceptor such as nitrate or oxygen ispresent.

In organisms such as E. coli, PEPCK operates in the gluconeogenic andATP-consuming direction from oxaloacetate towards phosphoenolpyruvate.It has been hypothesized that kinetic limitations of PEPCK of E. coliprevent it from effectively catalyzing the formation of oxaloacetatefrom PEP. PEP carboxylase (PPC), which does not generate ATP but isrequired for efficient growth, is naturally utilized by E. coli to formoxaloacetate from phosphoenolpyruvate. Therefore, three non native PEPCKenzymes (Table 26) were tested for their ability to complement growth ofa PPC mutant strain of E. coli in glucose minimal media.

TABLE 26 Sources of phosphoenolpyruvate carboxykinase sequences.Accession Number, GenBank Reference PEPCK Source Strain SequenceHaemophilus influenza NC_000907.1 Actinobacillus succinogenesYP_001343536.1 Mannheimia succiniciproducens YP_089485.1

Growth complementation studies involved plasmid based expression of thecandidate genes in Δppc mutant E. coli JW3978 obtained from the Keiocollection (Baba et al., Molecular Systems Biology 2:2006.0008 (2006)).The genes were cloned behind the PA1lacO-1 promoter in the expressionvectors pZA23 (medium copy) and pZE13 (high copy). These plasmids havebeen described previously (Lutz and Bujard, Nucleic Acids Res.25:1203-1210 (1997)), and their use in expression BDO pathway genes hasbeen described previously in WO2008115840.

Pre-cultures were grown aerobically in M9 minimal media with 4 g/Lglucose. All pre-cultures were supplemented with aspartate (2 mM) toprovide the Δppc mutants with a source for generating TCA cycleintermediates independent of PEPCK expression. M9 minimal media was alsoused in the test conditions with 4 g/L glucose, but no aspartate wasadded and IPTG was added to 0.5 mM. Table 27 shows the results of thegrowth complementation studies.

TABLE 27 Complementation of Δppc mutants with PEPCK from H. influenzae,A. succinogenes and M. succinoproducens when expressed from vectorspZA23 or pZE13. PEPCK Source Strain Vector Time (h) OD₆₀₀ H. influenzaepZA23BB 40 0.950 Δppc Control pZA23BB 40 0.038 A. succinogenes pZA23BB40 0.055 M. succinoproducens pZA23BB 40 0.214 A. succinogenes pZE13BB 400.041 M. succinoproducens pZE13BB 40 0.024 Δppc Control pZE13BB 40 0.042

Haemophilus influenza PEPCK was found to complement growth in Δppcmutant E. coli best among the genes that were tested in the plasmidbased screening. This gene was then integrated into the PPC locus ofwild-type E. coli (MG1655) using the SacB counter selection method withpRE118-V2 discussed above (Gay et al., J. Bacteriol. 153:1424-1431(1983)). PEPCK was integrated retaining the E. coli native PPC promoter,but utilizing the non-native PEPCK terminator. The sequence of thisregion following replacement of ppc by H. influenzae pepck is shown inFIG. 48. The pepck coding region is underlined.

Techniques for adaptive evolution were applied to improve the growthrate of the E. coli mutant (Δppc::H. inf pepCK). M9 minimal media with 4g/L glucose and 50 mM sodium bicarbonate was used to culture and evolvethis strain in an anaerobic environment. The high sodium bicarbonateconcentration was used to drive the equilibrium of the PEPCK reactiontoward oxaloacetate formation. To maintain exponential growth, theculture was diluted 2-fold whenever an OD600 of 0.5 was achieved. Afterabout 100 generations over 3 weeks of adaptive evolution, anaerobicgrowth rates improved from about 8 h to that of wild type, about 2 h.Following evolution, individual colonies were isolated, and growth inanaerobic bottles was compared to that of the initial mutant andwild-type strain (see FIG. 49). M9 medium with 4 g/L glucose and 50 mMsodium bicarbonate was used.

The ppc/pepck gene replacement procedure described above was thenrepeated, this time using the BDO-producing strains ECKh-432 (ΔadhEΔldhA ΔpflB ΔlpdA::K.p.lpdA322 Δmdh ΔarcA gltAR163L ΔackA fimD:: E. colisucCD, P. gingivalis sucD, P. gingivalis 4hbd fimD:: M. bovis sucA, C.kluyveri 4hbd) and ECKh-439 as the hosts. These strains contain the TCAcycle enhancements discussed above as well as the upstream pathwayintegrated in the chromosome. ECKh-439 is a derivative of ECKh-432 thathas the ackA gene deleted, which encodes acetate kinase. This deletionwas performed using the sacB counterselection method described above.

The Δppc::H. inf pepCK derivative of ECKh-439, called ECKh-453, was runin a fermentation. The downstream BDO pathway was supplied by pZS*13containing P. gingivalis Cat2 and C. beijerinckii Ald. This wasperformed with 1 L initial culture volume in 2 L Biostat B+ bioreactors(Sartorius) using M9 minimal medium supplemented with 20 g/L glucose and50 mM NaHCO₃. The temperature was controlled at 37° C., and the pH wascontrolled at 7.0 using 2 M NH₄OH or Na₂CO₃. Cells were grownaerobically to an OD600 of approximately 2, at which time the cultureswere induced with 0.2 mM IPTG. One hour following induction, the airflow rate was reduced to 0.01 standard liters per minute formicroaerobic conditions. The agitation rate was initially set at 700rpm. The aeration rate was gradually increased throughout thefermentation as the culture density increased. Concentrated glucosesolution was fed to maintain glucose concentration in the vessel between0.5 and 10 g/L. The product profile is shown in FIG. 50. The observedphenotype, in which BDO and acetate are produced in approximately aone-to-one molar ratio, is highly similar to that predicted in WO2009/023493 for design #439 (ADHEr, ASPT, LDH_D, MDH, PFLi). Thedeletion targeting the ASPT reaction was deemed unnecessary as thenatural flux through aspartate ammonia-lyase is low.

A key feature of OptKnock strains is that production of the metaboliteof interest is generally coupled to growth, and further, that,production should occur during exponential growth as well as instationary phase. The growth coupling potential of ECKh-432 and ECKh-453was evaluated by growth in microaerobic bottles with frequent samplingduring the exponential phase. M9 medium containing 4 g/L glucose andeither 10 mM NaHCO₃ (for ECKh-432) or 50 mM NaHCO₃ (for ECKh-453) wasused, and 0.2 mM IPTG was included from inoculation. 18G needles wereused for microaerobic growth of ECKh-432, while both 18G and 27G needleswere tested for ECKh-453. The higher gauge needles result in lessaeration. As shown in FIG. 51, ECKh-432 does not begin producing BDOuntil 5 g/L glucose has been consumed, corresponding to the onset ofstationary phase. ECKh-453 produces BDO more evenly throughout theexperiment. In addition, growth coupling improves as the aeration of theculture is reduced.

EXAMPLE XVII Integration of BDO Pathway Encoding Genes at SpecificIntegration Sites

This example describes integration of various BDO pathway genes into thefimD locus to provide more efficient expression and stability.

The entire upstream BDO pathway, leading to 4HB, has been integratedinto the E. coli chromosome at the fimD locus. The succinate branch ofthe upstream pathway was integrated into the E. coli chromosome usingthe λ red homologeous recombination method (Datsenko and Wanner, Proc.Natl. Acad. Sci. USA 97:6640-6645 (2000)). The recipient E. coli strainwas ECKh-422 (ΔadhE ΔldhA ΔpflB ΔlpdA::K.p.lpdA322 Δmdh ΔarcAgltAR163L). A polycistronic DNA fragment containing a promoter, thesucCD gene, the sucD gene and the 4hbd gene and a terminator sequencewas inserted into the AflIII site of the pKD3 plasmid. The followingprimers were used to amplify the operon together with thechloramphenicol marker from the plasmid. The underlined sequences arehomologeous to the target insertion site.

(SEQ ID NO: 41) 5′-GTTTGCACGCTATAGCTGAGGTTGTTGTCTTCCAGCAACGTACCGTATACAATAGGCGTATCACGAGGCCCTTTC-3′ (SEQ ID NO: 42)5′-GCTACAGCATGTCACACGATCTCAACGGTCGGATGACCAATCTGGCTGGTATGGGAATTAGCCATGGTCC-3′

Following DpnI treatment and DNA electrophoresis, the purified PCRproduct was used to transform E. coli strain harboring plasmid pKD46.The candidate strain was selected on plates containing chloramphenicol.Genomic DNA of the candidate strain was purified. The insertion sequencewas amplified and confirmed by DNA sequencing. Thechloramphenicol-resistant marker was removed from chromosome by flipase.The nucleotide sequence of the region after insertion and marker removalis shown in FIG. 52.

The alpha-ketoglutarate branch of the upstream pathway was integratedinto the chromosome by homologeous recombination. The plasmid used inthis modification was derived from vector pRE118-V2, as referenced inExample XIV, which contains a kanamycin-resistant gene, a gene encodingthe levansucrase (sacB) and a R6K conditional replication ori. Theintegration plasmid also contained a polycistronic sequence with apromoter, the sucA gene, the C. kluyveri 4hbd gene, and a terminatorbeing inserted between two 1.5-kb DNA fragments that are homologeous tothe flanking regions of the target insertion site. The resulting plasmidwas used to transform E. coli strain. The integration candidate wasselected on plates containing kanamycin. The correct integration sitewas verified by PCR. To resolve the antibiotic marker from thechromosome, the cells were selected for growth on medium containingsucrose. The final strain was verified by PCR and DNA sequencing. Thenucleotide sequence of the chromosomal region after insertion and markerremoval is shown in FIG. 53.

The resulting upstream pathway integration strain ECKh-432 wastransformed with a plasmid harboring the downstream pathway genes. Theconstruct was able to produce BDO from glucose in minimal medium (seeFIG. 54).

EXAMPLE XVIII Use of a Non-Phosphotransferase Sucrose Uptake System toReduce Pyruvate Byproduct Formation

This example describes the utilization of a non-phosphotransferase (PTS)sucrose uptake system to reduce pyruvate as a byproduct in theconversion of sucrose to BDO.

Strains engineered for the utilization of sucrose via aphosphotransferase (PTS) system produce significant amounts of pyruvateas a byproduct. Therefore, the use of a non-PTS sucrose system can beused to decrease pyruvate formation because the import of sucrose wouldnot be accompanied by the conversion of phosphoenolpyruvate (PEP) topyruvate. This will increase the PEP pool and the flux to oxaloacetatethrough PPC or PEPCK.

Insertion of a non-PTS sucrose operon into the rrnC region wasperformed. To generate a PCR product containing the non-PTS sucrosegenes flanked by regions of homology to the rrnC region, two oligos wereused to PCR amplify the csc genes from Mach1™ (Invitrogen, Carlsbad,Calif.). This strain is a descendent of W strain which is an E. colistrain known to be able to catabolize sucrose (Orencio-Trejo et al.,Biotechnology Biofuels 1:8 (2008)). The sequence was derived from E.coli W strain KO11 (accession AY314757) (Shukla et al., Biotechnol.Lett. 26:689-693 (2004)) and includes genes encoding a sucrose permease(cscB), D-fructokinase (cscK), sucrose hydrolase (cscA), and aLacI-related sucrose-specific repressor (cscR). The first 53 amino acidsof cscR was effectively removed by the placement of the AS primer. Thesequences of the oligos were: rrnC 23S del S-CSC 5′-TGT GAG TGA AAG TCACCT GCC TTA ATA TCT CAA AAC TCA TCT TCG GGT GAC GAA ATA TGG CGT GAC TCGATA C-3′ (SEQ ID NO:43) and rrnC 23S del AS-CSC 5′-TCT GTA TCA GGC TGAAAA TCT TCT CTC ATC CGC CAA AAC AGC TTC GGC GTT AAG ATG CGC GCT CAA GGAC-3′ (SEQ ID NO:44). Underlined regions indicate homology to the cscoperon, and bold sequence refers to sequence homology upstream anddownstream of the rrnC region. The sequence of the entire PCR product isshown in FIG. 55.

After purification, the PCR product was electroporated into MG1655electrocompetent cells which had been transformed with pRedET (tet) andprepared according to manufacturer's instructions(www.genebridges.com/gb/pdf/K001%20Q %20E %20BAC %20Modification%20Kit-version2.6-2007-screen.pdf). The PCR product was designed so thatit integrated into genome into the rrnC region of the chromosome. Iteffectively deleted 191 nucleotides upstream of rrlC (23S rRNA), all ofthe rrlC rRNA gene and 3 nucleotides downstream of rrlC and replaced itwith the sucrose operon, as shown in FIG. 56.

Transformants were grown on M9 minimal salts medium with 0.4% sucroseand individual colonies tested for presence of the sucrose operon bydiagnostic PCR. The entire rrnC::crcAKB region was transferred into theBDO host strain ECKh-432 by P1 transduction (Sambrook et al., MolecularCloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory,New York (2001), resulting in ECKh-463 (ΔadhE ΔldhA ΔpflBΔlpdA::K.p.lpdA322 Δmdh ΔarcA gltAR163L fimD:: E. coli sucCD, P.gingivalis sucD, P. gingivalis 4hbd fimD:: M. bovis sucA, C. kluyveri4hbd rrnC::cscAKB). Recombinants were selected by growth on sucrose andverified by diagnostic PCR.

ECKh-463 was transformed with pZS*13 containing P. gingivalis Cat2 andC. beijerinckii Ald to provide a complete BDO pathway. Cells werecultured in M9 minimal medium (6.78 g/L Na₂HPO₄, 3.0 g/L KH₂PO₄, 0.5 g/LNaCl, 1.0 g/L NH₄Cl, 1 mM MgSO₄, 0.1 mM CaCl₂) supplemented with 10 g/Lsucrose. 0.2 mM IPTG was present in the culture from the start.Anaerobic conditions were maintained using a bottle with 23G needle. Asa control, ECKh-432 containing the same plasmid was cultured on the samemedium, except with 10 g/L glucose instead of sucrose. FIG. 57 showsaverage product concentration, normalized to culture OD600, after 48hours of growth. The data is for 6 replicate cultures of each strain.This demonstrates that BDO production from ECKh-463 on sucrose issimilar to that of the parent strain on sucrose.

EXAMPLE XIX Summary of BDO Producing Strains

This example describes various BDO producting strains.

Table 28 summarizes various BDO producing strains disclosed above inExamples XII-XVIII.

TABLE 28 Summary of various BDO production strains. Host Strain # Strain# Host chromosome Host Description Plasmid-based 1 ΔldhA Single deletionE. coli sucCD, P. gingivalis derivative of E. coli sucD, P. gingivalis4hbd, P. gingivalis MG1655 Cat2, C. acetobutylicum AdhE2 2 AB3 ΔadhEΔldhA ΔpflB Succinate E. coli sucCD, P. gingivalis producing strain;sucD, P. gingivalis 4hbd, P. gingivalis derivative of E. coli Cat2, C.acetobutylicum MG1655 AdhE2 3 ECKh- ΔadhE ΔldhA ΔpflB Improvement of E.coli sucCD, P. gingivalis 138 ΔlpdA::K.p.lpdA322 lpdA to increase sucD,P. gingivalis 4hbd, P. gingivalis pyruvate Cat2, C. acetobutylicumdehydrogenase AdhE2 flux 4 ECKh- ΔadhE ΔldhA ΔpflB E. coli sucCD, P.gingivalis 138 ΔlpdA::K.p.lpdA322 sucD, P. gingivalis 4hbd, C.acetobutylicum buk1, C. acetobutylicum ptb, C. acetobutylicum AdhE2 5ECKh- ΔadhE ΔldhA ΔpflB Deletions in mdh E. coli sucCD, P. gingivalis401 ΔlpdA::K.p.lpdA322 Δmdh and arcA to direct sucD, P. gingivalis 4hbd,P. gingivalis ΔarcA flux through Cat2, C. acetobutylicum oxidative TCAAdhE2 cycle 6 ECKh- ΔadhE ΔldhA ΔpflB M. bovis sucA, E. coli sucCD, 401ΔlpdA::K.p.lpdA322 Δmdh P. gingivalis sucD, P. gingivalis ΔarcA 4hbd, P.gingivalis Cat2, C. acetobutylicum AdhE2 7 ECKh- ΔadhE ΔldhA ΔpflBMutation in citrate E. coli sucCD, P. gingivalis 422 ΔlpdA::K.p.lpdA322Δmdh synthase to sucD, P. gingivalis 4hbd, P. gingivalis ΔarcA gltAR163Limprove anaerobic Cat2, C. acetobutylicum activity AdhE2 8 ECKh- ΔadhEΔldhA ΔpflB M. bovis sucA, E. coli sucCD, 422 ΔlpdA::K.p.lpdA322 Δmdh P.gingivalis sucD, P. gingivalis ΔarcA gltAR163L 4hbd, P. gingivalis Cat2,C. acetobutylicum AdhE2 9 ECKh- ΔadhE ΔldhA ΔpflB M. bovis sucA, E. colisucCD, 422 ΔlpdA::K.p.lpdA322 Δmdh P. gingivalis sucD, P. gingivalisΔarcA gltAR163L 4hbd, P. gingivalis Cat2, C. beijerinckii Ald 10 ECKh-ΔadhE ΔldhA ΔpflB Succinate branch P. gingivalis Cat2, C. beijerinckii426 ΔlpdA::K.p.lpdA322 Δmdh of upstream Ald ΔarcA gltAR163L fimD:: E.coli pathway integrated sucCD, P. gingivalis sucD, P. gingivalis intoECKh-422 4hbd 11 ECKh- ΔadhE ΔldhA ΔpflB Succinate and P. gingivalisCat2, C. beijerinckii 432 ΔlpdA::K.p.lpdA322 Δmdh alpha-ketoglutarateAld ΔarcA gltAR163L fimD:: E. coli upstream pathway sucCD, P. gingivalissucD, P. gingivalis branches 4hbd fimD:: M. bovis integrated into sucA,C. kluyveri 4hbd ECKh-422 12 ECKh- ΔadhE ΔldhA ΔpflB C. acetobutylicumbuk1, C. acetobutylicum 432 ΔlpdA::K.p.lpdA322 Δmdh ptb, C. beijerinckiiΔarcA gltAR163L fimD:: E. coli Ald sucCD, P. gingivalis sucD, P.gingivalis 4hbd fimD:: M. bovis sucA, C. kluyveri 4hbd 13 ECKh- ΔadhEΔldhA ΔpflB Acetate kinase P. gingivalis Cat2, C. beijerinckii 439ΔlpdA::K.p.lpdA322 Δmdh deletion of ECKh- Ald ΔarcA gltAR163L ΔackAfimD:: 432 E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd fimD::M. bovis sucA, C. kluyveri 4hbd 14 ECKh- ΔadhE ΔldhA ΔpflB Acetatekinase P. gingivalis Cat2, C. beijerinckii 453 ΔlpdA::K.p.lpdA322 Δmdhdeletion and Ald ΔarcA gltAR163L ΔackA PPC/PEPCK Δppc::H.i.ppck fimD::E. coli replacement of sucCD, P. gingivalis sucD, P. gingivalis ECKh-4324hbd fimD:: M. bovis sucA, C. kluyveri 4hbd 15 ECKh- ΔadhE ΔldhA ΔpflBΔlpdA::fnr- Replacement of P. gingivalis Cat2, C. beijerinckii 456pflB6-K.p.lpdA322 Δmdh ΔarcA lpdA promoter Ald gltAR163L fimD:: E. coliwith anaerobic sucCD, P. gingivalis sucD, P. gingivalis promoter in 4hbdfimD:: M. bovis ECKh-432 sucA, C. kluyveri 4hbd 16 ECKh- ΔadhE ΔldhAΔpflB ΔlpdA:: Replacement of P. gingivalis Cat2, C. beijerinckii 455K.p.lpdA322 ΔpdhR:: fnr-pflB6 pdhR and aceEF Ald Δmdh ΔarcA gltAR163LfimD:: promoter with E. coli sucCD, P. gingivalis anaerobic sucD, P.gingivalis 4hbd fimD:: promoter in M. bovis sucA, C. kluyveri 4hbdECKh-432 17 ECKh- ΔadhE ΔldhA ΔpflB ΔlpdA:: Integration of C.beijerinckii Ald 459 K.p.lpdA322 Δmdh ΔarcA BK/PTB into gltAR163L fimD::E. coli ECKh-432 sucCD, P. gingivalis sucD, P. gingivalis 4hbd fimD:: M.bovis sucA, C. kluyveri 4hbd fimD:: C. acetobutylicum buk1, C.acetobutylicum ptb 18 ECKh- ΔadhE ΔldhA ΔpflB ΔlpdA:: C. beijerinckiiAld, G. thermoglucosidasius 459 K.p.lpdA322 Δmdh ΔarcA adh1 gltAR163LfimD:: E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd fimD:: M.bovis sucA, C. kluyveri 4hbd fimD:: C. acetobutylicum buk1, C.acetobutylicum ptb 19 ECKh- ΔadhE ΔldhA ΔpflB Non-PTS sucrose P.gingivalis Cat2, C. beijerinckii 463 ΔlpdA::K.p.lpdA322 Δmdh genesinserted into Ald ΔarcA gltAR163L fimD:: E. coli ECKh-432 sucCD, P.gingivalis sucD, P. gingivalis 4hbd fimD:: M. bovis sucA, C. kluyveri4hbd rrnC::cscAKB 20 ECKh- ΔadhE ΔldhA ΔpflB C. acetobutylicum buk1, C.acetobutylicum 463 ΔlpdA::K.p.lpdA322 Δmdh ptb, C. beijerinckii ΔarcAgltAR163L fimD:: E. coli Ald sucCD, P. gingivalis sucD, P. gingivalis4hbd fimD:: M. bovis sucA, C. kluyveri 4hbd rrnC::cscAKB

The strains summarized in Table 28 are as follows. Strain 1: Singledeletion derivative of E. coli MG1655, with deletion of endogenous ldhA;plasmid expression of E. coli sucCD, P. gingivalis sucD, P. gingivalis4hbd, P. gingivalis Cat2, C. acetobutylicum AdhE2. Strain 2: Host strainAB3, a succinate producing strain, derivative of E. coli MG1655, withdeletions of endogenous adhE ldhA pflB; plasmid expression of E. colisucCD, P. gingivalis sucD, P. gingivalis 4hbd, P. gingivalis Cat2, C.acetobutylicum AdhE2.

Strain 3: Host strain ECKh-138, deletion of endogenous adhE, ldhA, pflB,deletion of endogenous lpdA and chromosomal insertion of Klebsiellapneumoniae lpdA with a Glu354Lys mutation at the lpdA locus; plasmidexpression of E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd, P.gingivalis Cat2, C. acetobutylicum AdhE2; strain provides improvement oflpdA to increase pyruvate dehydrogenase flux. Strain 4: Host strainECKh-138, deletion of endogenous adhE, ldhA, pflB, and lpdA, chromosomalinsertion of Klebsiella pneumoniae lpdA with a Glu354Lys mutation;plasmid expression E. coli sucCD, P. gingivalis sucD, P. gingivalis4hbd, C. acetobutylicum buk1, C. acetobutylicum ptb, C. acetobutylicumAdhE2.

Strain 5: Host strain ECKh-401, deletion of endogenous adhE, ldhA, pflB,deletion of endogenous lpdA and chromosomal insertion of Klebsiellapneumoniae lpdA with a Glu354Lys mutation at the lpdA locus, deletion ofendogenous mdh and arcA; plasmid expression of E. coli sucCD, P.gingivalis sucD, P. gingivalis 4hbd, P. gingivalis Cat2, C.acetobutylicum AdhE2; strain has deletions in mdh and arcA to directflux through oxidative TCA cycle. Strain 6: host strain ECKh-401,deletion of endogenous adhE, ldhA, pflB, deletion of endogenous lpdA andchromosomal insertion of Klebsiella pneumoniae lpdA with a Glu354Lysmutation at the lpdA locus, deletion of endogenous mdh and arcA; plasmidexpression of M. bovis sucA, E. coli sucCD, P. gingivalis sucD, P.gingivalis 4hbd, P. gingivalis Cat2, C. acetobutylicum AdhE2.

Strain 7: Host strain ECKh-422, deletion of endogenous adhE, ldhA, pflB,deletion of endogenous lpdA and chromosomal insertion of Klebsiellapneumoniae lpdA with a Glu354Lys mutation at the lpdA locus, deletion ofendogenous mdh and arcA, chromosomal replacement of gltA with gltAArg163Leu mutant; plasmid expression of E. coli sucCD, P. gingivalissucD, P. gingivalis 4hbd, P. gingivalis Cat2, C. acetobutylicum AdhE2;strain has mutation in citrate synthase to improve anaerobic activity.Strain 8: strain ECKh-422, deletion of endogenous adhE, ldhA, pflB,deletion of endogenous lpdA and chromosomal insertion of Klebsiellapneumoniae lpdA with a Glu354Lys mutation at the lpdA locus, deletion ofendogenous mdh and arcA, chromosomal replacement of gltA with gltAArg163Leu mutant; plasmid expression of M. bovis sucA, E. coli sucCD, P.gingivalis sucD, P. gingivalis 4hbd, P. gingivalis Cat2, C.acetobutylicum AdhE2. Strain 9: host strain ECKh-422, deletion ofendogenous adhE, ldhA, pflB, deletion of endogenous lpdA and chromosomalinsertion of Klebsiella pneumoniae lpdA with a Glu354Lys mutation at thelpdA locus, deletion of endogenous mdh and arcA, chromosomal replacementof gltA with gltA Arg163Leu mutant; plasmid expression of M. bovis sucA,E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd, P. gingivalisCat2, C. beijerinckii Ald.

Strain 10: host strain ECKh-426, deletion of endogenous adhE, ldhA,pflB, deletion of endogenous lpdA and chromosomal insertion ofKlebsiella pneumoniae lpdA with a Glu354Lys mutation at the lpdA locus,deletion of endogenous mdh and arcA, chromosomal replacement of gltAwith gltA Arg163Leu mutant, chromosomal insertion at the fimD locus ofE. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd; plasmidexpression of P. gingivalis Cat2, C. beijerinckii Ald; strain hassuccinate branch of upstream pathway integrated into strain ECKh-422 atthe fimD locus.

Strain 11: host strain ECKh-432, deletion of endogenous adhE, ldhA,pflB, deletion of endogenous lpdA and chromosomal insertion ofKlebsiella pneumoniae lpdA with a Glu354Lys mutation at the lpdA locus,deletion of endogenous mdh and arcA, chromosomal replacement of gltAwith gltA Arg163Leu mutant, chromosomal insertion at the fimD locus ofE. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd, chromosomalinsertion at the fimD locus of M. bovis sucA, C. kluyveri 4hbd; plasmidexpression of P. gingivalis Cat2, C. beijerinckii Ald; strain hassuccinate and alpha-ketoglutarate upstream pathway branches integratedinto ECKh-422. Strain 12: host strain ECKh-432, deletion of endogenousadhE, ldhA, pflB, deletion of endogenous lpdA and chromosomal insertionof Klebsiella pneumoniae lpdA with a Glu354Lys mutation at the lpdAlocus, deletion of endogenous mdh and arcA, chromosomal replacement ofgltA with gltA Arg163Leu mutant, chromosomal insertion at the fimD locusof E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd, chromosomalinsertion at the fimD locus of M. bovis sucA, C. kluyveri 4hbd; plasmidexpression of C. acetobutylicum buk1, C. acetobutylicum ptb, C.beijerinckii Ald.

Strain 13: host strain ECKh-439, deletion of endogenous adhE, ldhA,pflB, deletion of endogenous lpdA and chromosomal insertion ofKlebsiella pneumoniae lpdA with a Glu354Lys mutation at the lpdA locus,deletion of endogenous mdh and arcA, chromosomal replacement of gltAwith gltA Arg163Leu mutant, deletion of endogenous ackA, chromosomalinsertion at the fimD locus of E. coli sucCD, P. gingivalis sucD, P.gingivalis 4hbd, chromosomal insertion at the fimD locus of M. bovissucA, C. kluyveri 4hbd; plasmid expression of P. gingivalis Cat2, C.beijerinckii Ald; strain has acetate kinase deletion in strain ECKh-432.Strain 14: host strain ECKh-453, deletion of endogenous adhE, ldhA,pflB, deletion of endogenous lpdA and chromosomal insertion ofKlebsiella pneumoniae lpdA with a Glu354Lys mutation at the lpdA locus,deletion of endogenous mdh and arcA, chromosomal replacement of gltAwith gltA Arg163Leu mutant, deletion of endogenous ackA, deletion ofendogenous ppc and insertion of Haemophilus influenza ppck at the ppclocus, chromosomal insertion at the fimD locus of E. coli sucCD, P.gingivalis sucD, P. gingivalis 4hbd, chromosomal insertion at the fimDlocus of M. bovis sucA, C. kluyveri 4hbd; plasmid expression of P.gingivalis Cat2, C. beijerinckii Ald; strain has acetate kinase deletionand PPC/PEPCK replacement in strain ECKh-432.

Strain 15: host strain ECKh-456, deletion of endogenous adhE, ldhA,pflB, deletion of endogenous lpdA and chromosomal insertion ofKlebsiella pneumoniae lpdA with a Glu354Lys mutation at the lpdA locus,deletion of endogenous mdh and arcA, chromosomal replacement of gltAwith gltA Arg163Leu mutant, chromosomal insertion at the fimD locus ofE. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd, chromosomalinsertion at the fimD locus of M. bovis sucA, C. kluyveri 4hbd,replacement of lpdA promoter with fnr binding site, pflB-p6 promoter andRBS of pflB; plasmid expression of P. gingivalis Cat2, C. beijerinckiiAld; strain has replacement of lpdA promoter with anaerobic promoter instrain ECKh-432. Strain 16: host strain ECKh-455, deletion of endogenousadhE, ldhA, pflB, deletion of endogenous lpdA and chromosomal insertionof Klebsiella pneumoniae lpdA with a Glu354Lys mutation at the lpdAlocus, deletion of endogenous mdh and arcA, chromosomal replacement ofgltA with gltA Arg163Leu mutant, chromosomal insertion at the fimD locusof E. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd, chromosomalinsertion at the fimD locus of M. bovis sucA, C. kluyveri 4hbd,replacement of pdhR and aceEF promoter with fnr binding site, pflB-p6promoter and RBS of pflB; plasmid expression of P. gingivalis Cat2, C.beijerinckii Ald; strain has replacement of pdhR and aceEF promoter withanaerobic promoter in ECKh-432.

Strain 17: host strain ECKh-459, deletion of endogenous adhE, ldhA,pflB, deletion of endogenous lpdA and chromosomal insertion ofKlebsiella pneumoniae lpdA with a Glu354Lys mutation at the lpdA locus,deletion of endogenous mdh and arcA, chromosomal replacement of gltAwith gltA Arg163Leu mutant, chromosomal insertion at the fimD locus ofE. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd, chromosomalinsertion at the fimD locus of M. bovis sucA, C. kluyveri 4hbd,chromosomal insertion at the fimD locus of C. acetobutylicum buk1, C.acetobutylicum ptb; plasmid expression of C. beijerinckii Ald; strainhas integration of BK/PTB into strain ECKh-432. Strain 18: host strainECKh-459, deletion of endogenous adhE, ldhA, pflB, deletion ofendogenous lpdA and chromosomal insertion of Klebsiella pneumoniae lpdAwith a Glu354Lys mutation at the lpdA locus, deletion of endogenous mdhand arcA, chromosomal replacement of gltA with gltA Arg163Leu mutant,chromosomal insertion at the fimD locus of E. coli sucCD, P. gingivalissucD, P. gingivalis 4hbd, chromosomal insertion at the fimD locus of M.bovis sucA, C. kluyveri 4hbd, chromosomal insertion at the fimD locus ofC. acetobutylicum buk1, C. acetobutylicum ptb; plasmid expression of C.beijerinckii Ald, G. thermoglucosidasius adh1.

Strain 19: host strain ECKh-463, deletion of endogenous adhE, ldhA,pflB, deletion of endogenous lpdA and chromosomal insertion ofKlebsiella pneumoniae lpdA with a Glu354Lys mutation at the lpdA locus,deletion of endogenous mdh and arcA, chromosomal replacement of gltAwith gltA Arg163Leu mutant, chromosomal insertion at the fimD locus ofE. coli sucCD, P. gingivalis sucD, P. gingivalis 4hbd, chromosomalinsertion at the fimD locus of M. bovis sucA, C. kluyveri 4hbd,insertion at the rrnC locus of non-PTS sucrose operon genes sucrosepermease (cscB), D-fructokinase (cscK), sucrose hydrolase (cscA), and aLacI-related sucrose-specific repressor (cscR); plasmid expression of P.gingivalis Cat2, C. beijerinckii Ald; strain has non-PTS sucrose genesinserted into strain ECKh-432. Strain 20: host strain ECKh-463 deletionof endogenous adhE, ldhA, pflB, deletion of endogenous lpdA andchromosomal insertion of Klebsiella pneumoniae lpdA with a Glu354Lysmutation at the lpdA locus, deletion of endogenous mdh and arcA,chromosomal replacement of gltA with gltA Arg163Leu mutant, chromosomalinsertion at the fimD locus of E. coli sucCD, P. gingivalis sucD, P.gingivalis 4hbd, chromosomal insertion at the fimD locus of M. bovissucA, C. kluyveri 4hbd, insertion at the rrnC locus of non-PTS sucroseoperon; plasmid expression of C. acetobutylicum buk1, C. acetobutylicumptb, C. beijerinckii Ald.

In addition to the BDO producing strains disclosed herein, includingthose disclosed in Table 28, it is understood that additionalmodifications can be incorporated that further increase production ofBDO and/or decrease undesirable byproducts. For example, a BDO producingstrain, or a strain of Table 28, can incorporate additional knockouts tofurther increase the production of BDO or decrease an undesirablebyproduct. Exemplary knockouts have been described previously (see U.S.publication 2009/0047719). Such knockout strains include, but are notlimited to, one ore more genes selected from ADHEr, NADH6; ADHEr, PPCK;ADHEr, SUCD4; ADHEr, ATPS4r; ADHEr, FUM; ADHEr, MDH; ADHEr, PFLi, PPCK;ADHEr, PFLi, SUCD4; ADHEr, ACKr, NADH6; ADHEr, NADH6, PFLi; ADHEr, ASPT,MDH; ADHEr, NADH6, PPCK; ADHEr, PPCK, THD2; ADHEr, ATPS4r, PPCK; ADHEr,MDH, THD2; ADHEr, FUM, PFLi; ADHEr, PPCK, SUCD4; ADHEr, GLCpts, PPCK;ADHEr, GLUDy, MDH; ADHEr, GLUDy, PPCK; ADHEr, FUM, PPCK; ADHEr, MDH,PPCK; ADHEr, FUM, GLUDy; ADHEr, FUM, HEX1; ADHEr, HEX1, PFLi; ADHEr,HEX1, THD2; ADHEr, FRD2, LDH_D, MDH; ADHEr, FRD2, LDH_D, ME2; ADHEr,MDH, PGL, THD2; ADHEr, G6PDHy, MDH, THD2; ADHEr, PFLi, PPCK, THD2;ADHEr, ACKr, AKGD, ATPS4r; ADHEr, GLCpts, PFLi, PPCK; ADHEr, ACKr,ATPS4r, SUCOAS; ADHEr, GLUDy, PFLi, PPCK; ADHEr, ME2, PFLi, SUCD4;ADHEr, GLUDy, PFLi, SUCD4; ADHEr, ATPS4r, LDH_D, SUCD4; ADHEr, FUM,HEX1, PFLi; ADHEr, MDH, NADH6, THD2; ADHEr, ATPS4r, MDH, NADH6; ADHEr,ATPS4r, FUM, NADH6; ADHEr, ASPT, MDH, NADH6; ADHEr, ASPT, MDH, THD2;ADHEr, ATPS4r, GLCpts, SUCD4; ADHEr, ATPS4r, GLUDy, MDH; ADHEr, ATPS4r,MDH, PPCK; ADHEr, ATPS4r, FUM, PPCK; ADHEr, ASPT, GLCpts, MDH; ADHEr,ASPT, GLUDy, MDH; ADHEr, ME2, SUCD4, THD2; ADHEr, FUM, PPCK, THD2;ADHEr, MDH, PPCK, THD2; ADHEr, GLUDy, MDH, THD2; ADHEr, HEX1, PFLi,THD2; ADHEr, ATPS4r, G6PDHy, MDH; ADHEr, ATPS4r, MDH, PGL; ADHEr, ACKr,FRD2, LDH_D; ADHEr, ACKr, LDH_D, SUCD4; ADHEr, ATPS4r, FUM, GLUDy;ADHEr, ATPS4r, FUM, HEX1; ADHEr, ATPS4r, MDH, THD2; ADHEr, ATPS4r, FRD2,LDH_D; ADHEr, ATPS4r, MDH, PGDH; ADHEr, GLCpts, PPCK, THD2; ADHEr,GLUDy, PPCK, THD2; ADHEr, FUM, HEX1, THD2; ADHEr, ATPS4r, ME2, THD2;ADHEr, FUM, ME2, THD2; ADHEr, GLCpts, GLUDy, PPCK; ADHEr, ME2, PGL,THD2; ADHEr, G6PDHy, ME2, THD2; ADHEr, ATPS4r, FRD2, LDH_D, ME2; ADHEr,ATPS4r, FRD2, LDH_D, MDH; ADHEr, ASPT, LDH_D, MDH, PFLi; ADHEr, ATPS4r,GLCpts, NADH6, PFLi; ADHEr, ATPS4r, MDH, NADH6, PGL; ADHEr, ATPS4r,G6PDHy, MDH, NADH6; ADHEr, ACKr, FUM, GLUDy, LDH_D; ADHEr, ACKr, GLUDy,LDH_D, SUCD4; ADHEr, ATPS4r, G6PDHy, MDH, THD2; ADHEr, ATPS4r, MDH, PGL,THD2; ADHEr, ASPT, G6PDHy, MDH, PYK; ADHEr, ASPT, MDH, PGL, PYK; ADHEr,ASPT, LDH_D, MDH, SUCOAS; ADHEr, ASPT, FUM, LDH_D, MDH; ADHEr, ASPT,LDH_D, MALS, MDH; ADHEr, ASPT, ICL, LDH_D, MDH; ADHEr, FRD2, GLUDy,LDH_D, PPCK; ADHEr, FRD2, LDH_D, PPCK, THD2; ADHEr, ACKr, ATPS4r, LDH_D,SUCD4; ADHEr, ACKr, ACS, PPC, PPCK; ADHEr, GLUDy, LDH_D, PPC, PPCK;ADHEr, LDH_D, PPC, PPCK, THD2; ADHEr, ASPT, ATPS4r, GLCpts, MDH; ADHEr,G6PDHy, MDH, NADH6, THD2; ADHEr, MDH, NADH6, PGL, THD2; ADHEr, ATPS4r,G6PDHy, GLCpts, MDH; ADHEr, ATPS4r, GLCpts, MDH, PGL; ADHEr, ACKr,LDH_D, MDH, SUCD4.

Table 29 shows the reactions of corresponding genes to be knocked out ofa host organism such as E. coli. The corresponding metabolitecorresponding to abbreviations in Table 29 are shown in Table 30.

TABLE 29 Corresponding genes to be knocked out to prevent a particularreaction from occurring in E. coli. Genes Encoding the ReactionEnzyme(s) Catalyzing Abbreviation Reaction Stoichiometry* Each Reaction&ACKr [c]: ac + atp <==> actp + adp (b3115 or b2296 or b1849) ACS [c]:ac + atp + coa --> accoa + amp + ppi b4069 ACt6 ac[p] + h[p] <==>ac[c] + h[c] Non-gene associated ADHEr [c]: etoh + nad <==> acald + h +nadh (b0356 or b1478 or b1241) [c]: acald + coa + nad <==> accoa + h +nadh (b1241 or b0351) AKGD [c]: akg + coa + nad --> co2 + nadh + succoa(b0116 and b0726 and b0727) ASNS2 [c]: asp-L + atp + nh4 --> amp +asn-L + h + ppi b3744 ASPT [c]: asp-L --> fum + nh4 b4139 ATPS4radp[c] + (4) h[p] + pi[c] <==> atp[c] + (3) h[c] + h2o[c] (((b3736 andb3737 and b3738) and (b3731 and b3732 and b3733 and b3734 and b3735)) or((b3736 and b3737 and b3738) and (b3731 and b3732 and b3733 and b3734and b3735) and b3739)) CBMK2 [c]: atp + co2 + nh4 <==> adp + cbp + (2) h(b0521 or b0323 or b2874) EDA [c]: 2ddg6p --> g3p + pyr b1850 ENO [c]:2pg <==> h2o + pep b2779 FBA [c]: fdp <==> dhap + g3p (b2097 or b2925 orb1773) FBP [c]: fdp + h2o --> f6p + pi (b4232 or b3925) FDH2 for[p] +(2) h[c] + q8[c] --> co2[c] + h[p] + q8h2[c] ((b3892 and b3893 for[p] +(2) h[c] + mqn8[c] --> co2[c] + h[p] + mql8[c] and b3894) or (b1474 andb1475 and b1476)) FRD2 [c]: fum + mql8 --> mqn8 + succ (b4151 and b4152[c]: 2dmmql8 + fum --> 2dmmq8 + succ and b4153 and b4154) FTHFD [c]:10fthf + h2o --> for + h + thf b1232 FUM [c]: fum + h2o <==> mal-L(b1612 or b4122 or b1611) G5SD [c]: glu5p + h + nadph --> glu5sa +nadp + pi b0243 G6PDHy [c]: g6p + nadp <==> 6pgl + h + nadph b1852GLCpts glc-D[p] + pep[c] --> g6p[c] + pyr[c] ((b2417 and b1101 and b2415and b2416) or (b1817 and b1818 and b1819 and b2415 and b2416) or (b2417and b1621 and b2415 and b2416)) GLU5K [c]: atp + glu-L --> adp + glu5pb0242 GLUDy [c]: glu-L + h2o + nadp <==> akg + h + nadph + nh4 b1761GLYCL [c]: gly + nad + thf --> co2 + mlthf + nadh + nh4 (b2904 and b2903and b2905 and b0116) HEX1 [c]: atp + glc-D --> adp + g6p + h b2388 ICL[c]: icit --> glx + succ b4015 LDH_D [c]: lac-D + nad <==> h + nadh +pyr (b2133 or b1380) MALS [c]: accoa + glx + h2o --> coa + h + mal-L(b4014 or b2976) MDH [c]: mal-L + nad <==> h + nadh + oaa b3236 ME2 [c]:mal-L + nadp --> co2 + nadph + pyr b2463 MTHFC [c]: h2o + methf <==>10fthf + h b0529 NADH12 [c]: h + mqn8 + nadh --> mql8 + nad b1109 [c]:h + nadh + q8 --> nad + q8h2 [c]: 2dmmq8 + h + nadh --> 2dmmql8 + nadNADH6 (4) h[c] + nadh[c] + q8[c] --> (3) h[p] + nad[c] + q8h2[c] (b2276and b2277 (4) h[c] + mqn8[c] + nadh[c] --> (3) h[p] + mql8[c] + andb2278 and b2279 nad[c] and b2280 and b2281 2dmmq8[c] + (4) h[c] +nadh[c] --> 2dmmql8[c] + (3) and b2282 and b2283 h[p] + nad[c] and b2284and b2285 and b2286 and b2287 and b2288) PFK [c]: atp + f6p --> adp +fdp + h (b3916 or b1723) PFLi [c]: coa + pyr --> accoa + for (((b0902and b0903) and b2579) or (b0902 and b0903) or (b0902 and b3114) or(b3951 and b3952)) PGDH [c]: 6pgc + nadp --> co2 + nadph + ru5p-D b2029PGI [c]: g6p <==> f6p b4025 PGL [c]: 6pgl + h2o --> 6pgc + h b0767 PGM[c]: 2pg <==> 3pg (b3612 or b4395 or b0755) PPC [c]: co2 + h2o + pep -->h + oaa + pi b3956 PPCK [c]: atp + oaa --> adp + co2 + pep b3403 PRO1z[c]: fad + pro-L --> 1pyr5c + fadh2 + h b1014 PYK [c]: adp + h + pep -->atp + pyr b1854 or b1676) PYRt2 h[p] + pyr[p] <==> h[c] + pyr[c]Non-gene associated RPE [c]: ru5p-D <==> xu5p-D (b4301 or b3386) SO4t2so4[e] <==> so4[p] (b0241 or b0929 or b1377 or b2215) SUCD4 [c]: q8 +succ --> fum + q8h2 (b0721 and b0722 and b0723 and b0724) SUCOAS [c]:atp + coa + succ <==> adp + pi + succoa (b0728 and b0729) SULabcatp[c] + h2o[c] + so4[p] --> adp[c] + h[c] + pi[c] + ((b2422 and b2425so4[c] and b2424 and b2423) or (b0763 and b0764 and b0765) or (b2422 andb2424 and b2423 and b3917)) TAL [c]: g3p + s7p <==> e4p + f6p (b2464 orb0008) THD2 (2) h[p] + nadh[c] + nadp[c] --> (2) h[c] + nad[c] + (b1602and b1603) nadph[c] THD5 [c]: nad + nadph --> nadh + nadp (b3962 or(b1602 and b1603)) TPI [c]: dhap <==> g3p b3919

TABLE 30 Metabolite names corresponding to abbreviations used in Table29. Metabolite Abbreviation Metabolite Name 10fthf10-Formyltetrahydrofolate 1pyr5c 1-Pyrroline-5-carboxylate 2ddg6p2-Dehydro-3-deoxy-D-gluconate 6-phosphate 2dmmq8 2-Demethylmenaquinone 82dmmql8 2-Demethylmenaquinol 8 2pg D-Glycerate 2-phosphate 3pg3-Phospho-D-glycerate 6pgc 6-Phospho-D-gluconate 6pgl6-phospho-D-glucono-1,5-lactone ac Acetate acald Acetaldehyde accoaAcetyl-CoA actp Acetyl phosphate adp ADP akg 2-Oxoglutarate amp AMPasn-L L-Asparagine asp-L L-Aspartate atp ATP cbp Carbamoyl phosphate co2CO2 coa Coenzyme A dhap Dihydroxyacetone phosphate e4p D-Erythrose4-phosphate etoh Ethanol f6p D-Fructose 6-phosphate fad Flavin adeninedinucleotide oxidized fadh2 Flavin adenine dinucleotide reduced fdpD-Fructose 1,6-bisphosphate for Formate fum Fumarate g3p Glyceraldehyde3-phosphate g6p D-Glucose 6-phosphate glc-D D-Glucose glu5p L-Glutamate5-phosphate glu5sa L-Glutamate 5-semialdehyde glu-L L-Glutamate glxGlyoxylate gly Glycine h H+ h2o H2O icit Isocitrate lac-D D-Lactatemal-L L-Malate methf 5,10-Methenyltetrahydrofolate mlthf5,10-Methylenetetrahydrofolate mql8 Menaquinol 8 mqn8 Menaquinone 8 nadNicotinamide adenine dinucleotide nadh Nicotinamide adeninedinucleotide - reduced nadp Nicotinamide adenine dinucleotide phosphatenadph Nicotinamide adenine dinucleotide phosphate - reduced nh4 Ammoniumoaa Oxaloacetate pep Phosphoenolpyruvate pi Phosphate ppi Diphosphatepro-L L-Proline pyr Pyruvate q8 Ubiquinone-8 q8h2 Ubiquinol-8 ru5p-DD-Ribulose 5-phosphate s7p Sedoheptulose 7-phosphate so4 Sulfate succSuccinate succoa Succinyl-CoA thf 5,6,7,8-Tetrahydrofolate xu5p-DD-Xylulose 5-phosphate

EXAMPLE XX Exemplary Pathways for Producing BDO

This example describes exemplary pathways to produce 4-hydroxybutanal(4-HBal) and/or BDO using a carboxylic acid reductase as a BDO pathwayenzyme.

An exemplary pathway for production of BDO includes use of an NAD+ orNADP+ aryl-aldehyde dehydrogenase (E.C.: 1.2.1.29 and 1.2.1.30) toconvert 4-hydroxybutyrate to 4-hydroxybutanal and an alcoholdehydrogenase to convert 4-hydroxybutanal to 1,4-butanediol.4-Hydroxybutyrate can be derived from the tricarboxylic acid cycleintermediates succinyl-CoA and/or alpha-ketoglutarate as shown in FIG.58.

Aryl-Aldehyde Dehydrogenase (or Carboxylic Acid Reductase). Anaryl-aldehyde dehydrogenase, or equivalently a carboxylic acidreductase, can be found in Nocardia iowensis. Carboxylic acid reductasecatalyzes the magnesium, ATP and NADPH-dependent reduction of carboxylicacids to their corresponding aldehydes (Venkitasubramanian et al., J.Biol. Chem. 282:478-485 (2007)) and is capable of catalyzing theconversion of 4-hydroxybutyrate to 4-hydroxybutanal. This enzyme,encoded by car, was cloned and functionally expressed in E. coli(Venkitasubramanian et al., J. Biol. Chem. 282:478-485 (2007)).Expression of the npt gene product improved activity of the enzyme viapost-transcriptional modification. The npt gene encodes a specificphosphopantetheine transferase (PPTase) that converts the inactiveapo-enzyme to the active holo-enzyme. The natural substrate of thisenzyme is vanillic acid, and the enzyme exhibits broad acceptance ofaromatic and aliphatic substrates (Venkitasubramanian et al., inBiocatalysis in the Pharmaceutical and Biotechnology Industires, ed.R.N. Patel, Chapter 15, pp. 425-440, CRC Press LLC, Boca Raton, Fla.(2006)).

GenBank Accession Gene name GI No. No. Organism car 40796035 AAR91681.1Nocardia iowensis (sp. NRRL 5646) npt 14848891 ABI83656.1 Nocardiaiowensis (sp. NRRL 5646)

Additional car and npt genes can be identified based on sequencehomology.

GenBank Accession Gene name GI No. No. Organism fadD9 121638475YP_978699.1 Mycobacterium bovis BCG BCG_2812c 121638674 YP_978898.1Mycobacterium bovis BCG nfa20150 54023983 YP_118225.1 Nocardia farcinicaIFM 10152 nfa40540 54026024 YP_120266.1 Nocardia farcinica IFM 10152SGR_6790 182440583 YP_001828302.1 Streptomyces griseus subsp. griseusNBRC 13350 SGR_665 182434458 YP_001822177.1 Streptomyces griseus subsp.griseus NBRC 13350 MSMEG_2956 YP_887275.1 YP_887275.1 Mycobacteriumsmegmatis MC2 155 MSMEG_5739 YP_889972.1 118469671 Mycobacteriumsmegmatis MC2 155 MSMEG_2648 YP_886985.1 118471293 Mycobacteriumsmegmatis MC2 155 MAP1040c NP_959974.1 41407138 Mycobacterium aviumsubsp. paratuberculosis K-10 MAP2899c NP_961833.1 41408997 Mycobacteriumavium subsp. paratuberculosis K-10 MMAR_2117 YP_001850422.1 183982131Mycobacterium marinum M MMAR_2936 YP_001851230.1 183982939 Mycobacteriummarinum M MMAR_1916 YP_001850220.1 183981929 Mycobacterium marinum MTpauDRAFT_33060 ZP_04027864.1 227980601 Tsukamurella paurometabola DSM20162 TpauDRAFT_20920 ZP_04026660.1 ZP_04026660.1 Tsukamurellapaurometabola DSM 20162 CPCC7001_1320 ZP_05045132.1 254431429 CyanobiumPCC7001 DDBDRAFT_0187729 XP_636931.1 66806417 Dictyostelium discoideumAX4

An additional enzyme candidate found in Streptomyces griseus is encodedby the griC and griD genes. This enzyme is believed to convert3-amino-4-hydroxybenzoic acid to 3-amino-4-hydroxybenzaldehyde asdeletion of either griC or griD led to accumulation of extracellular3-acetylamino-4-hydroxybenzoic acid, a shunt product of3-amino-4-hydroxybenzoic acid metabolism (Suzuki, et al., J. Antibiot.60(6):380-387 (2007)). Co-expression of griC and griD with SGR_665, anenzyme similar in sequence to the Nocardia iowensis npt, can bebeneficial.

GenBank Accession Gene name GI No. No. Organism griC 182438036YP_001825755.1 Streptomyces griseus subsp. griseus NBRC 13350 griD182438037 YP_001825756.1 Streptomyces griseus subsp. griseus NBRC 13350

An enzyme with similar characteristics, alpha-aminoadipate reductase(AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in somefungal species. This enzyme naturally reduces alpha-aminoadipate toalpha-aminoadipate semialdehyde. The carboxyl group is first activatedthrough the ATP-dependent formation of an adenylate that is then reducedby NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizesmagnesium and requires activation by a PPTase. Enzyme candidates for AARand its corresponding PPTase are found in Saccharomyces cerevisiae(Morris et al., Gene 98:141-145 (1991)), Candida albicans (Guo et al.,Mol. Genet. Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe(Ford et al., Curr. Genet. 28:131-137 (1995)). The AAR from S. pombeexhibited significant activity when expressed in E. coli (Guo et al.,Yeast 21:1279-1288 (2004)). The AAR from Penicillium chrysogenum acceptsS-carboxymethyl-L-cysteine as an alternate substrate, but did not reactwith adipate, L-glutamate or diaminopimelate (Hijarrubia et al., J.Biol. Chem. 278:8250-8256 (2003)). The gene encoding the P. chrysogenumPPTase has not been identified to date.

GenBank Accession Gene name GI No. No. Organism LYS2 171867 AAA34747.1Saccharomyces cerevisiae LYS5 1708896 P50113.1 Saccharomyces cerevisiaeLYS2 2853226 AAC02241.1 Candida albicans LYS5 28136195 AAO26020.1Candida albicans Lys1p 13124791 P40976.3 Schizosaccharomyces pombe Lys7p1723561 Q10474.1 Schizosaccharomyces pombe Lys2 3282044 CAA74300.1Penicillium chrysogenum

There are several advantages of using carboxylic acid reductase for BDOproduction. There are at least two advantages of forming4-hydroxybutanal from 4-hydroxybutyrate via a carboxylic acid reductasecompared to forming 4-hydroxybutanal from an activated version of4-hydroxybutyrate (for example, 4-hydroxybutyryl-CoA,4-hydroxybutyryl-Pi) via an acyl-CoA or acyl-phosphate reductase. First,the formation of gamma-butyrolactone (GBL) as a byproduct is greatlyreduced. It is believed that the activated versions of 4-hydroxybutyratecyclize to GBL more readily than unactivated 4-hydroxybutyrate. The useof carboxylic acid reductase eliminates the need to pass through a freeactivated 4-hydroxybutyrate intermediate, thus reducing the formation ofGBL as a byproduct accompanying BDO production. Second, the formation ofethanol as a byproduct is greatly reduced. Ethanol is often formed invarying amounts when an aldehyde- or an alcohol-forming4-hydroxybutyryl-CoA reductase is used to convert 4-hydroxybutyryl-CoAto 4-hydroxybutanal or 1,4-butanediol, respectively. This is becausemost, if not all, aldehyde- or alcohol-forming 4-hydroxybutyryl-CoAreductases can accept acetyl-CoA as a substrate in addition to4-hydroxybutyryl-CoA. Aldehyde-forming enzymes, for example, oftencatalyze the conversion of acetyl-CoA to acetaldehyde, which issubsequently reduced to ethanol by native or non-native alcoholdehydrogenases. Alcohol-forming 4-hydroxybutyryl-CoA reductases thataccept acetyl-CoA as a substrate will convert acetyl-CoA directly toethanol. It appears that carboxylic acid reductase enzymes have far lessactivity on acetyl-CoA than aldehyde- or alcohol-forming acyl-CoAreductase enzymes, and thus their application for BDO production resultsin minimal ethanol byproduct formation (see below).

EXAMPLE XXI Biosynthesis of 1,4-Butanediol Using a Carboxylic AcidReductase Enzyme

This example describes the generation of a microbial organism thatproduces 1,4-butanediol using a carboxylic acid reductase enzyme.

Escherichia coli is used as a target organism to engineer the pathwayfor 1,4-butanediol synthesis described in FIG. 58. E. coli provides agood host for generating a non-naturally occurring microorganism capableof producing 1,4-butanediol. E. coli is amenable to genetic manipulationand is known to be capable of producing various products, like ethanol,acetic acid, formic acid, lactic acid, and succinic acid, effectivelyunder various oxygenation conditions.

Integration of 4-Hydroxybutyrate Pathway Genes into Chromosome:Construction of ECKh-432. The carboxylic acid reductase enzyme wasexpressed in a strain of E. coli designated ECKh-432 whose constructionis described in Example XVII. This strain contained the components ofthe BDO pathway, leading to 4HB, integrated into the chromosome of E.coli at the fimD locus.

As described in Example XVII, the succinate branch of the upstreampathway was integrated into the E. coli chromosome using the λ redhomologeous recombination method (Datsenko and Wanner, Proc. Natl. Acad.Sci. USA 97:6640-6645 (2000)). A polycistronic DNA fragment containing apromoter, the sucCD gene of Escherichia coli encoding succinyl-CoAligase, the sucD gene of Porphyromonas gingivalis encoding succinyl-CoAreductase (aldehyde forming) (step A of FIG. 58), the 4hbd gene ofPorphyromonas gingivalis encoding 4-hydroxybutyrate dehydrogenase (stepC of FIG. 58), and a terminator sequence was inserted into the AflIIIsite of the pKD3 plasmid.

As described in Example XVII, the alpha-ketoglutarate branch of theupstream pathway was integrated into the chromosome by homologeousrecombination. The plasmid used in this modification was pRE118-V2(pRE118 (ATCC87693) deleted of the oriT and IS sequences), whichcontains a kanamycin-resistant gene, a gene encoding the levansucrase(sacB) and a R6K conditional replication ori. The integration plasmidalso contained a polycistronic sequence with a promoter, the sucA genefrom Mycobacterium bovis encoding alpha-ketoglutarate decarboxylase(step B of FIG. 58), the Clostridium kluyveri 4hbd gene encoding4-hydroxybutyrate dehydrogenase (step C of FIG. 58), and a terminatorbeing inserted between two 1.5-kb DNA fragments that are homologous tothe flanking regions of the target insertion site. The resulting plasmidwas used to transform E. coli strain. The integration candidate wasselected on plates containing kanamycin. The correct integration sitewas verified by PCR. To resolve the antibiotic marker from thechromosome, the cells were selected for growth on medium containingsucrose. The final strain was verified by PCR and DNA sequencing.

The recipient E. coli strain was ECKh-422 (ΔadhE ΔldhA ΔpflBΔlpdA::K.p.lpdA322 Δmdh ΔarcA gltAR163L) whose construction is describedin Example XV. ECKh-422 contains a mutation gltAR163L leading toNADH-insensitivity of citrate synthase encoded by gltA. It furthercontains an NADH-insensitive version of the lpdA gene from Klebsiellapneumonia integrated into the chromosome as described below.

Replacement of the native lpdA was replaced with a NADH-insensitive lpdAfrom Klebsiella pneumonia, as described in Example XIV. The resultingvector was designated pRE118-V2 (see FIG. 34).]

Cloning and Expression of Carboxylic Acid Reductase and PPTase. Togenerate an E. coli strain engineered to produce 1,4-butanediol, nucleicacids encoding a carboxylic acid reductase and phosphopantetheinetransferase are expressed in E. coli using well known molecular biologytechniques (see, for example, Sambrook, supra, 2001; Ausubel supra,1999). In particular, the car (AAR91681.1) and npt (ABI83656.1) geneswere cloned into the pZS*13 vector (Expressys, Ruelzheim, Germany) underthe PA1/lacO promoter. The car gene (GNM_720) was cloned by PCR fromNocardia genomic DNA. Its nucleic acid and protein sequences are shownin FIGS. 59A and 59B, respectively.

A codon-optimized version of the npt gene (GNM_721) was synthesized byGeneArt (Regensburg, Germany). Its nucleic acid and protein sequencesare shown in FIGS. 60A and 60B, respectively. The resulting vector fromcloning GNM_720 and GNM_721 into pZS*13 is shown in FIG. 61.

The plasmid was transformed into ECKh-432 to express the proteins andenzymes required for 1,4-butanediol production. Alternate versions ofthe plasmid containing only GNM_720 and only GNM_721 were alsoconstructed.

Demonstration of 1,4-BDO Production Using Carboxylic Acid Reductase.Functional expression of the 1,4-butanediol pathway was demonstratedusing E. coli whole-cell culture. A single colony of E. coli ECKh-432transformed with the pZS*13 plasmid containing both GNM_720 and GNM_721was inoculated into 5 mL of LB medium containing appropriateantibiotics. Similarly, single colonies of E. coli ECKh-432 transformedwith the pZS*13 plasmids containing either GNM_720 or GNM_721 wereinoculated into additional 5 mL aliquots of LB medium containingappropriate antibiotics. Ten mL micro-aerobic cultures were started byinoculating fresh minimal in vivo conversion medium (see below)containing the appropriate antibiotics with 1% of the first cultures.

Recipe of the minimal in vivo conversion medium (for 1000 mL) is asfollows:

final concentration 1M MOPS/KOH buffer 40 mM Glucose (40%) 1% 10XM9salts solution 1X MgSO4 (1M) 1 mM trace minerals (×1000) 1X 1M NaHCO3 10mM

Microaerobic conditions were established by initially flushing cappedanaerobic bottles with nitrogen for 5 minutes, then piercing the septumwith an 18G needle following inoculation. The needle was kept in thebottle during growth to allow a small amount of air to enter thebottles. Protein expression was induced with 0.2 mM IPTG when theculture reached mid-log growth phase. This is considered: time=0 hr. Theculture supernatants were analyzed for BDO, 4HB, and other by-productsas described above and in WO2008115840 (see Table 31).

TABLE 31 Production of BDO, 4-HB and other products in various strains.mM Strain pZS*13S OD600 OD600 PA SA LA 4HB BDO GBL ETOH_(Enz) ECKh-432720 0.420 2.221 6.36 0.00 0.10 7.71 3.03 0.07 >LLOQ ECKh-432 721 0.3232.574 1.69 0.00 0.00 12.60 0.00 0.00 >LLOQ ECKh-432 720/721 0.378 2.4691.70 0.00 0.01 4.23 9.16 0.24 1.52 PA = pyruvate, SA = succinate, LA =lactate, 4HB = 4-hydroxybutyrate, BDO = 1,4-butanediol, GBL =gamma-butyrolactone, Etoh = ethanol, LLOQ = lower limit ofquantification

These results demonstrate that the carboxylic acid reductase gene,GNM_720, is required for BDO formation in ECKh-432 and its effectivenessis increased when co-expressed with the PPTase, GNM_721. GBL and ethanolwere produced in far smaller quantities than BDO in the strainsexpressing GNM_720 by itself or in combination with GNM_721.

Additional Pathways to BDO Employing Carboxylic Acid Reductase. It isexpected that carboxylic acid reductase can function as a component ofmany pathways to 1,4-butanediol from the TCA cycle metabolites:succinate, succinyl-CoA, and alpha-ketoglutarate. Several of thesepathways are disclosed in FIG. 62. All routes can lead to theoreticalBDO yields greater than or equal to 1 mol/mol assuming glucose as thecarbon source. Similar high theoretical yields can be obtained fromadditional substrates including sucrose, xylose, arabinose, synthesisgas, among many others. It is expected that the expression of carboxylicacid reductase alone or in combination with PPTase (that is, to catalyzesteps F and D of FIG. 62) is sufficient for 1,4-butanediol productionfrom succinate provided that sufficient endogenous alcohol dehydrogenaseactivity is present to catalyze steps C and E of FIG. 62. Candidateenzymes for steps A through Z of FIG. 62 are described in section XXIII.

EXAMPLE XXII Pathways to Putrescine that Employ Carboxylic AcidReductase

This example describes exemplary putrescine pathways utilizingcarboxylic acid reductase.

Putrescine, also known as 1,4-diaminobutane or butanediamine, is anorganic chemical compound of the formula NH₂(CH₂)₄NH₂. It can be reactedwith adipic acid to yield the polyamide Nylon-4,6, which is marketed byDSM (Heerlen, Netherlands) under the trade name Stanyl™ Putrescine isnaturally produced, for example, by the natural breakdown of amino acidsin living and dead organisms. E. coli has been engineered to produceputrescine by overexpressing the native ornithine biosynthetic machineryas well as an ornithine decarboxylase (Qian, et al., Biotechnol. Bioeng.104(4):651-662 (2009)).

FIG. 63 describes a number of additional biosynthetic pathways leadingto the production of putrescine from succinate, succinyl-CoA, oralpha-ketoglutarate and employing a carboxylic acid reductase. Note thatnone of these pathways require formation of an activated version of4-aminobutyrate such as 4-aminobutyryl-CoA, which can be reduced by anacyl-CoA reductase to 4-aminobutanal but also can readily cyclize to itslactam, 2-pyrrolidinone (Ohsugi, et al., J. Biol. Chem. 256:7642-7651(1981)). All routes can lead to theoretical putrescine yields greaterthan or equal to 1 mol/mol assuming glucose as the carbon source.Similar high theoretical yields can be obtained from additionalsubstrates including sucrose, xylose, arabinose, synthesis gas, amongmany others. Candidate enzymes for steps A through U of FIG. 63 aredescribed in Example XXIII.

EXAMPLE XXIII Exemplary Enzymes for Production of C4 Compounds

This example describes exemplary enzymes for production of C4 compoundssuch as 1,4-butanediol, 4-hydroxybutanal and putrescine.

Enzyme Classes. All transformations depicted in FIGS. 58, 62 and 63 fallinto the general categories of transformations shown in Table 32. Thisexample describes a number of biochemically characterized genes in eachcategory. Specifically listed are genes that can be applied to catalyzethe appropriate transformations in FIGS. 58, 62 and 63 when cloned andexpressed. The first three digits of each label correspond to the firstthree Enzyme Commission number digits which denote the general type oftransformation independent of substrate specificity.

TABLE 32 Classes of Enzyme Transformations Depicted in FIGS. 58, 62 and63. LABEL FUNCTION 1.1.1.a Oxidoreductase (oxo to alcohol) 1.1.1.cOxidoreductase (2 step, acyl-CoA to alcohol) 1.2.1.b Oxidoreductase(acyl-CoA to aldehyde) 1.2.1.c Oxidoreductase (2-oxo acid to acyl-CoA,decarboxylation) 1.2.1.d Oxidoreductase (phosphonate reductase) 1.2.1.eAcid reductase 1.4.1.a Oxidoreductase (aminating) 2.3.1.aAcyltransferase (transferring phosphate group to CoA) 2.6.1.aAminotransferase 2.7.2.a Phosphotransferase (carboxy acceptor) 2.8.3.aCoA transferase 3.1.2.a CoA hydrolase 4.1.1.a Carboxy-lyase 6.2.1.a CoAsynthetase1.1.1.a Oxidoreductase (Oxo to Alcohol)

Aldehyde to Alcohol. Three transformations described in FIGS. 58, 62 and63 involve the conversion of an aldehyde to alcohol. These are4-hydroxybutyrate dehydrogenase (step C, FIGS. 58 and 62),1,4-butanediol dehydrogenase (step E, FIGS. 58 and 62), and5-hydroxy-2-pentanoic acid (step Y, FIG. 62). Exemplary genes encodingenzymes that catalyze the conversion of an aldehyde to alcohol, that is,alcohol dehydrogenase or equivalently aldehyde reductase, include alrAencoding a medium-chain alcohol dehydrogenase for C2-C14 (Tani et al.Appl. Environ. Microbiol. 66:5231-5235 (2000)), ADH2 from Saccharomycescerevisiae (Atsumi et al. Nature 451:86-89 (2008)), yqhD from E. coli,which has preference for molecules longer than C(3) (Sulzenbacher et al.J. Mol. Biol. 342:489-502 (2004)), and bdh I and bdh II from C.acetobutylicum, which converts butyryaldehyde into butanol (Walter etal. J. Bacteriol. 174:7149-7158 (1992)). The protein sequences for eachof exemplary gene products can be found using the following GenBankaccession numbers:

Gene Accession No. GI No. Organism alrA BAB12273.1 9967138 Acinetobactersp. Strain M-1 ADH2 NP_014032.1 6323961 Saccharymyces cerevisiae yqhDNP_417484.1 16130909 Escherichia coli bdh I NP_349892.1 15896543Clostridium acetobutylicum bdh II NP_349891.1 15896542 Clostridiumacetobutylicum

Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity (EC1.1.1.61) also fall into this category. Such enzymes have beencharacterized in Ralstonia eutropha (Bravo et al. J. Forensic Sci.49:379-387 (2004)), Clostridium kluyveri (Wolff et al., Protein Expr.Pur 6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz et al. J.Biol. Chem. 278:41552-41556 (2003)).

Gene Accession No. GI No. Organism 4hbd YP_726053.1 113867564 Ralstoniaeutropha H16 4hbd EDK35022.1 146348486 Clostridium kluyveri DSM 555 4hbdQ94B07 75249805 Arabidopsis thaliana

The adh1 gene from Geobacillus thermoglucosidasius M10EXG (Jeon et al.,J. Biotechnol. 135:127-133 (2008)) was shown to exhibit high activity onboth 4-hydroxybutanal and butanal (see above). Thus this enzyme exhibits1,4-butanediol dehydrogenase activity.

Gene Accession No. GI No. Organism adh1 AAR91477.1 40795502 Geobacillusthermoglucosidasius M10EXG

Another exemplary enzyme is 3-hydroxyisobutyrate dehydrogenase, whichcatalyzes the reversible oxidation of 3-hydroxyisobutyrate tomethylmalonate semialdehyde. This enzyme participates in valine, leucineand isoleucine degradation and has been identified in bacteria,eukaryotes, and mammals. The enzyme encoded by P84067 from Thermusthermophilus HB8 has been structurally characterized (Lokanath et al. J.Mol. Biol. 352:905-17 (2005)). The reversibility of the human3-hydroxyisobutyrate dehydrogenase was demonstrated usingisotopically-labeled substrate (Manning et al., Biochem. J. 231:481-484(1985)). Additional genes encoding this enzyme include 3hidh in Homosapiens (Hawes et al., Methods Enzymol. 324:218-228 (2000)) andOryctolagus cuniculus (Chowdhury et al., Biosci. Biotechnol. Biochem.60:2043-2047 (1996); Hawes et al. Methods Enzymol. 324:218-228 (2000)),mmsb in Pseudomonas aeruginosa, and dhat in Pseudomonas putida (Aberhartet al., J. Chem. Soc. [Perkin 1] 6:1404-1406 (1979); Chowdhury et al.,Biosci. Biotechnol Biochem. 67:438-441 (2003); Chowdhury et al., Biosci.Biotechnol. Biochem. 60:2043-2047 (1996)).

Gene Accession No. GI No. Organism P84067 P84067 75345323 Thermusthermophilus mmsb P28811.1 127211 Pseudomonas aeruginosa dhat Q59477.12842618 Pseudomonas putida 3hidh P31937.2 12643395 Homo sapiens 3hidhP32185.1 416872 Oryctolagus cuniculus

Several 3-hydroxyisobutyrate dehydrogenase enzymes have also been shownto convert malonic semialdehyde to 3-hydroxyproprionic acid (3-HP).Three gene candidates exhibiting this activity are mmsB from Pseudomonasaeruginosa PAO1(62), mmsB from Pseudomonas putida KT2440 (Liao et al.,US Publication 2005/0221466) and mmsB from Pseudomonas putida E23(Chowdhury et al., Biosci. Biotechnol. Biochem. 60:2043-2047 (1996)). Anenzyme with 3-hydroxybutyrate dehydrogenase activity in Alcaligenesfaecalis M3A has also been identified (Gokam et al., U.S. Pat. No.7,393,676; Liao et al., US Publication No. 2005/0221466). Additionalgene candidates from other organisms including Rhodobacter spaeroidescan be inferred by sequence similarity.

Gene Accession No. GI No. Organism mmsB AAA25892.1 151363 Pseudomonasaeruginosa mmsB NP_252259.1 15598765 Pseudomonas aeruginosa PAO1 mmsBNP_746775.1 26991350 Pseudomonas putida KT2440 mmsB JC7926 60729613Pseudomonas putida E23 orfB1 AAL26884 16588720 Rhodobacter spaeroides

The conversion of malonic semialdehyde to 3-HP can also be accomplishedby two other enzymes, NADH-dependent 3-hydroxypropionate dehydrogenaseand NADPH-dependent malonate semialdehyde reductase. An NADH-dependent3-hydroxypropionate dehydrogenase is thought to participate inbeta-alanine biosynthesis pathways from propionate in bacteria andplants (Rathinasabapathi, J. Plant Pathol. 159:671-674 (2002); Stadtman,J. Am. Chem. Soc. 77:5765-5766 (1955)). This enzyme has not beenassociated with a gene in any organism to date. NADPH-dependent malonatesemialdehyde reductase catalyzes the reverse reaction in autotrophicCO₂-fixing bacteria. Although the enzyme activity has been detected inMetallosphaera sedula, the identity of the gene is not known (Alber etal. J. Bacteriol. 188:8551-8559 (2006)).

1.1.1.c Oxidoreductase (2 Step, Acyl-CoA to Alcohol).

Steps S and W of FIG. 62 depict bifunctional reductase enzymes that canform 4-hydroxybutyrate and 1,4-butanediol, respectively. Exemplary2-step oxidoreductases that convert an acyl-CoA to alcohol include thosethat transform substrates such as acetyl-CoA to ethanol (for example,adhE from E. coli (Kessler et al., FEBS. Lett. 281:59-63 (1991)) andbutyryl-CoA to butanol (for example, adhE2 from C. acetobutylicum(Fontaine et al., J. Bacteriol. 184:821-830 (2002)). The C.acetobutylicum adhE2 gene was shown to convert 4-hydroxybutyryl-CoA to1,4-butanediol (see above). In addition to reducing acetyl-CoA toethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides hasbeen shown to oxidize the branched chain compound isobutyraldehyde toisobutyryl-CoA (Kazahaya et al. J., Gen. Appl. Microbiol. 18:43-55(1972); Koo et al., Biotechnol. Lett. 27:505-510 (2005)).

Gene Accession No. GI No. Organism adhE NP_415757.1 16129202 Escherichiacoli adhE2 AAK09379.1 12958626 Clostridium acetobutylicum adhEAAV66076.1 55818563 Leuconostoc mesenteroides

Another exemplary enzyme can convert malonyl-CoA to 3-HP. AnNADPH-dependent enzyme with this activity has characterized inChloroflexus aurantiacus, where it participates in the3-hydroxypropionate cycle (Hugler et al., J. Bacteriol. 184:2404-2410(2002); Strauss and Fuchs, Eur. J. Biochem. 215:633-643 (1993)). Thisenzyme, with a mass of 300 kDa, is highly substrate-specific and showslittle sequence similarity to other known oxidoreductases (Hugler etal., J. Bacteriol. 184:2404-2410 (2002)). No enzymes in other organismshave been shown to catalyze this specific reaction; however there isbioinformatic evidence that other organisms may have similar pathways(Klatt et al., Environ. Microbiol. 9:2067-2078 (2007)). Enzymecandidates in other organisms including Roseiflexus castenholzii,Erythrobacter sp. NAP1 and marine gamma proteobacterium HTCC2080 can beinferred by sequence similarity.

Gene Accession No. GI No. Organism mcr AAS20429.1 42561982 Chloroflexusaurantiacus Rcas_2929 YP_001433009.1 156742880 Roseiflexus castenholziiNAP1_ 02720 ZP_01039179.1 85708113 Erythrobacter sp. NAP1 MGP2080_00535ZP_01626393.1 119504313 marine gamma proteobacterium HTCC2080

Longer chain acyl-CoA molecules can be reduced by enzymes such as thejojoba (Simmondsia chinensis) FAR, which encodes an alcohol-formingfatty acyl-CoA reductase. Its overexpression in E. coli resulted in FARactivity and the accumulation of fatty alcohol (Metz et al., PlantPhysiol. 122:635-644 2000)).

Gene Accession No. GI No. Organism FAR AAD38039.1 5020215 Simmondsiachinensis1.2.1.b Oxidoreductase (Acyl-CoA to Aldehyde).

Step A of FIGS. 58, 62 and 63 involves the conversion of succinyl-CoA tosuccinate semialdehyde by an aldehyde forming succinyl-CoA reductase.Step Q of FIG. 62 depicts the conversion of 4-hydroxybutyryl-CoA to4-hydroxybutanal by an aldehyde-forming 4-hydroxybutyryl-CoA reductase.Several acyl-CoA dehydrogenases are capable of reducing an acyl-CoA toits corresponding aldehyde. Exemplary genes that encode such enzymesinclude the Acinetobacter calcoaceticus acyl encoding a fatty acyl-CoAreductase (Reiser and Somerville, J. Bacteriol. 179:2969-2975 (1997)),the Acinetobacter sp. M-1 fatty acyl-CoA reductase (Ishige et al., Appl.Environ. Microbiol. 68:1192-1195 (2002)), and a CoA- and NADP-dependentsuccinate semialdehyde dehydrogenase encoded by the sucD gene inClostridium kluyveri (Sohling and Gottschalk, J. Bacteriol. 178:871-80(1996); Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996)). SucDof P. gingivalis is another aldehyde-forming succinyl-CoA reductase(Takahashi et al., J. Bacteriol. 182:4704-4710 (2000)). The enzymeacylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG,is yet another as it has been demonstrated to oxidize and acylateacetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde andformaldehyde (Powlowski et al., J. Bacteriol. 175:377-385 (1993)). Inaddition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhEin Leuconostoc mesenteroides has been shown to oxidize the branchedchain compound isobutyraldehyde to isobutyryl-CoA (Koo et al.,Biotechnol. Lett. 27:505-510 (2005)). Butyraldehyde dehydrogenasecatalyzes a similar reaction, conversion of butyryl-CoA tobutyraldehyde, in solventogenic organisms such as Clostridiumsaccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol. Biochem.71:58-68 (2007)).

Gene Accession No. GI No. Organism acr1 YP_047869.1 50086359Acinetobacter calcoaceticus acr1 AAC45217 1684886 Acinetobacter baylyiacr1 BAB85476.1 18857901 Acinetobacter sp. Strain M-1 sucD P38947.1730847 Clostridium kluyveri sucD NP_904963.1 34540484 Porphyromonasgingivalis bphG BAA03892.1 425213 Pseudomonas sp adhE AAV66076.155818563 Leuconostoc mesenteroides bld AAP42563.1 31075383 Clostridiumsaccharoperbutylacetonicum

An additional enzyme type that converts an acyl-CoA to its correspondingaldehyde is malonyl-CoA reductase, which transforms malonyl-CoA tomalonic semialdehyde. Malonyl-CoA reductase is a key enzyme inautotrophic carbon fixation via the 3-hydroxypropionate cycle inthermoacidophilic archael bacteria (Berg et al., Science 318:1782-1786(2007); Thauer, Science 318:1732-1733 (2007)). The enzyme utilizes NADPHas a cofactor and has been characterized in Metallosphaera andSulfolobus spp (Alber et al., J. Bacteriol. 188:8551-8559 (2006); Hugleret al., J. Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded byMsed_0709 in Metallosphaera sedula (Alber et al., J. Bacteriol.188:8551-8559 (2006); Berg et al., Science 318:1782-1786 (2007)). A geneencoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned andheterologously expressed in E. coli (Alber et al., J. Bacteriol.188:8551-8559 (2006)). Although the aldehyde dehydrogenase functionalityof these enzymes is similar to the bifunctional dehydrogenase fromChloroflexus aurantiacus, there is little sequence similarity. Bothmalonyl-CoA reductase enzyme candidates have high sequence similarity toaspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reductionand concurrent dephosphorylation of aspartyl-4-phosphate to aspartatesemialdehyde. Additional gene candidates can be found by sequencehomology to proteins in other organisms including Sulfolobussolfataricus and Sulfolobus acidocaldarius. Yet another candidate forCoA-acylating aldehyde dehydrogenase is the ald gene from Clostridiumbeijerinckii (Toth et al., Appl. Environ. Microbiol. 65:4973-4980(1999)). This enzyme has been reported to reduce acetyl-CoA andbutyryl-CoA to their corresponding aldehydes. This gene is very similarto eutE that encodes acetaldehyde dehydrogenase of Salmonellatyphimurium and E. coli (Toth et al., Appl. Environ. Microbiol.65:4973-4980 (1999)).

Gene Accession No. GI No. Organism Msed_0709 YP_001190808.1 146303492Metallosphaera sedula mcr NP_378167.1 15922498 Sulfolobus tokodaii asd-2NP_343563.1 15898958 Sulfolobus solfataricus Saci_2370 YP_256941.170608071 Sulfolobus acidocaldarius Ald AAT66436 49473535 Clostridiumbeijerinckii eutE AAA80209 687645 Salmonella typhimurium eutE P774452498347 Escherichia coli1.2.1.c Oxidoreductase (2-Oxo Acid to Acyl-CoA, Decarboxylation).

Step AA in FIG. 62 depicts the conversion of 5-hydroxy-2-oxopentanoicacid to 4-hydroxybutyryl-CoA. Candidate enzymes for this transformationinclude 1) branched-chain 2-keto-acid dehydrogenase, 2)alpha-ketoglutarate dehydrogenase, and 3) the pyruvate dehydrogenasemultienzyme complex (PDHC). These enzymes are multi-enzyme complexesthat catalyze a series of partial reactions which result in acylatingoxidative decarboxylation of 2-keto-acids. Each of the 2-keto-aciddehydrogenase complexes occupies key positions in intermediarymetabolism, and enzyme activity is typically tightly regulated (Fries etal. Biochemistry 42:6996-7002 (2003)). The enzymes share a complex butcommon structure composed of multiple copies of three catalyticcomponents: alpha-ketoacid decarboxylase (E1), dihydrolipoamideacyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). The E3component is shared among all 2-keto-acid dehydrogenase complexes in anorganism, while the E1 and E2 components are encoded by different genes.The enzyme components are present in numerous copies in the complex andutilize multiple cofactors to catalyze a directed sequence of reactionsvia substrate channeling. The overall size of these dehydrogenasecomplexes is very large, with molecular masses between 4 and 10 millionDa (that is, larger than a ribosome).

Activity of enzymes in the 2-keto-acid dehydrogenase family is normallylow or limited under anaerobic conditions in E. coli. Increasedproduction of NADH (or NADPH) could lead to a redox-imbalance, and NADHitself serves as an inhibitor to enzyme function. Engineering effortshave increased the anaerobic activity of the E. coli pyruvatedehydrogenase complex (Kim et al. Appl. Environ. Microbiol. 73:1766-1771(2007); Kim et al. J. Bacteriol. 190:3851-3858) 2008); Zhou et al.Biotechnol. Lett. 30:335-342 (2008)). For example, the inhibitory effectof NADH can be overcome by engineering an H322Y mutation in the E3component (Kim et al. J. Bacteriol. 190:3851-3858 (2008)). Structuralstudies of individual components and how they work together in complexprovide insight into the catalytic mechanisms and architecture ofenzymes in this family (Aevarsson et al. Nat. Struct. Biol. 6:785-792(1999); Zhou et al. Proc. Natl. Acad. Sci. U.S.A. 98:14802-14807(2001)). The substrate specificity of the dehydrogenase complexes variesin different organisms, but generally branched-chain keto-aciddehydrogenases have the broadest substrate range.

Alpha-ketoglutarate dehydrogenase (AKGD) converts alpha-ketoglutarate tosuccinyl-CoA and is the primary site of control of metabolic fluxthrough the TCA cycle (Hansford, R. G. Curr. Top. Bioenerg. 10:217-278(1980)). Encoded by genes sucA, sucB and lpd in E. coli, AKGD geneexpression is downregulated under anaerobic conditions and during growthon glucose (Park et al. Mol. Microbiol. 15:473-482 (1995)). Although thesubstrate range of AKGD is narrow, structural studies of the catalyticcore of the E2 component pinpoint specific residues responsible forsubstrate specificity (Knapp et al. J. Mol. Biol. 280:655-668 (1998)).The Bacillus subtilis AKGD, encoded by odhAB (E1 and E2) and pdhD (E3,shared domain), is regulated at the transcriptional level and isdependent on the carbon source and growth phase of the organism(Resnekov et al. Mol. Gen. Genet. 234:285-296 (1992)). In yeast, theLPD1 gene encoding the E3 component is regulated at the transcriptionallevel by glucose (Roy and Dawes J. Gen. Microbiol. 133:925-933 (1987)).The E1 component, encoded by KGD1, is also regulated by glucose andactivated by the products of HAP2 and HAP3 (Repetto and Tzagoloff Mol.Cell Biol. 9:2695-2705 (1989)). The AKGD enzyme complex, inhibited byproducts NADH and succinyl-CoA, is well-studied in mammalian systems, asimpaired function of has been linked to several neurological diseases(Tretter and dam-Vizi Philos. Trans. R. Soc. Lond B Biol. Sci.360:2335-2345 (2005)).

Gene Accession No. GI No. Organism sucA NP_415254.1 16128701 Escherichiacoli str. K12 substr. MG1655 sucB NP_415255.1 16128702 Escherichia colistr. K12 substr. MG1655 lpd NP_414658.1 16128109 Escherichia coli str.K12 substr. MG1655 odhA P23129.2 51704265 Bacillus subtilis odhBP16263.1 129041 Bacillus subtilis pdhD P21880.1 118672 Bacillus subtilisKGD1 NP_012141.1 6322066 Saccharomyces cerevisiae KGD2 NP_010432.16320352 Saccharomyces cerevisiae LPD1 NP_116635.1 14318501 Saccharomycescerevisiae

Branched-chain 2-keto-acid dehydrogenase complex (BCKAD), also known as2-oxoisovalerate dehydrogenase, participates in branched-chain aminoacid degradation pathways, converting 2-keto acids derivatives ofvaline, leucine and isoleucine to their acyl-CoA derivatives and CO₂.The complex has been studied in many organisms including Bacillussubtilis (Wang et al. Eur. J. Biochem. 213:1091-1099 (1993)), Rattusnorvegicus (Namba et al. J. Biol. Chem. 244:4437-4447 (1969)) andPseudomonas putida (Sokatch J. Bacteriol. 148:647-652 (1981)). InBacillus subtilis the enzyme is encoded by genes pdhD (E3 component),bfmBB (E2 component), bfmBAA and bfmBAB (E1 component) (Wang et al. Eur.J. Biochem. 213:1091-1099 (1993)). In mammals, the complex is regulatedby phosphorylation by specific phosphatases and protein kinases. Thecomplex has been studied in rat hepatocites (Chicco et al. J. Biol.Chem. 269:19427-19434 (1994)) and is encoded by genes Bckdha (E1 alpha),Bckdhb (E1 beta), Dbt (E2), and Dld (E3). The E1 and E3 components ofthe Pseudomonas putida BCKAD complex have been crystallized (Aevarssonet al. Nat. Struct. Biol. 6:785-792 (1999); Mattevi Science255:1544-1550 (1992)) and the enzyme complex has been studied (Sokatchet al. J. Bacteriol. 148:647-652 (1981)). Transcription of the P. putidaBCKAD genes is activated by the gene product of bkdR (Hester et al. Eur.J. Biochem. 233:828-836 (1995)). In some organisms including Rattusnorvegicus (Paxton et al. Biochem. J. 234:295-303 (1986)) andSaccharomyces cerevisiae (Sinclair et al. Biochem. Mol. Biol. Int.31:911-922 (1993)), this complex has been shown to have a broadsubstrate range that includes linear oxo-acids such as 2-oxobutanoateand alpha-ketoglutarate, in addition to the branched-chain amino acidprecursors. The active site of the bovine BCKAD was engineered to favoralternate substrate acetyl-CoA (Meng and Chuang, Biochemistry33:12879-12885 (1994)).

Gene Accession No. GI No. Organism bfmBB NP_390283.1 16079459 Bacillussubtilis bfmBAA NP_390285.1 16079461 Bacillus subtilis bfmBABNP_390284.1 16079460 Bacillus subtilis pdhD P21880.1 118672 Bacillussubtilis lpdV P09063.1 118677 Pseudomonas putida bkdB P09062.1 129044Pseudomonas putida bkdA1 NP_746515.1 26991090 Pseudomonas putida bkdA2NP_746516.1 26991091 Pseudomonas putida Bckdha NP_036914.1 77736548Rattus norvegicus Bckdhb NP_062140.1 158749538 Rattus norvegicus DbtNP_445764.1 158749632 Rattus norvegicus Dld NP_955417.1 40786469 Rattusnorvegicus

The pyruvate dehydrogenase complex, catalyzing the conversion ofpyruvate to acetyl-CoA, has also been extensively studied. In the E.coli enzyme, specific residues in the E1 component are responsible forsubstrate specificity (Bisswanger, H. J Biol. Chem. 256:815-822 (1981);Bremer, J. Eur. J Biochem. 8:535-540 (1969); Gong et al. J Biol. Chem.275:13645-13653 (2000)). As mentioned previously, enzyme engineeringefforts have improved the E. coli PDH enzyme activity under anaerobicconditions (Kim et al. Appl. Environ. Microbiol. 73:1766-1771 (2007);Kim J. Bacteriol. 190:3851-3858 (2008); Zhou et al. Biotechnol. Lett.30:335-342 (2008)). In contrast to the E. coli PDH, the B. subtiliscomplex is active and required for growth under anaerobic conditions(Nakano J. Bacteriol. 179:6749-6755 (1997)). The Klebsiella pneumoniaePDH, characterized during growth on glycerol, is also active underanaerobic conditions (Menzel et al. J. Biotechnol. 56:135-142 (1997)).Crystal structures of the enzyme complex from bovine kidney (Zhou et al.Proc. Natl. Acad. Sci. U.S.A. 98:14802-14807 (2001)) and the E2catalytic domain from Azotobacter vinelandii are available (Mattevi etal. Science 255:1544-1550 (1992)). Some mammalian PDH enzymes complexescan react on alternate substrates such as 2-oxobutanoate, althoughcomparative kinetics of Rattus norvegicus PDH and BCKAD indicate thatBCKAD has higher activity on 2-oxobutanoate as a substrate (Paxton etal. Biochem. J. 234:295-303 (1986)).

Gene Accession No. GI No. Organism aceE NP_414656.1 16128107 Escherichiacoli str. K12 substr. MG1655 aceF NP_414657.1 16128108 Escherichia colistr. K12 substr. MG1655 lpd NP_414658.1 16128109 Escherichia coli str.K12 substr. MG1655 pdhA P21881.1 3123238 Bacillus subtilis pdhB P21882.1129068 Bacillus subtilis pdhC P21883.2 129054 Bacillus subtilis pdhDP21880.1 118672 Bacillus subtilis aceE YP_001333808.1 152968699Klebsiella pneumonia MGH78578 aceF YP_001333809.1 152968700 Klebsiellapneumonia MGH78578 lpdA YP_001333810.1 152968701 Klebsiella pneumoniaMGH78578 Pdha1 NP_001004072.2 124430510 Rattus norvegicus Pdha2NP_446446.1 16758900 Rattus norvegicus Dlat NP_112287.1 78365255 Rattusnorvegicus Dld NP_955417.1 40786469 Rattus norvegicus

As an alternative to the large multienzyme 2-keto-acid dehydrogenasecomplexes described above, some anaerobic organisms utilize enzymes inthe 2-ketoacid oxidoreductase family (OFOR) to catalyze acylatingoxidative decarboxylation of 2-keto-acids. Unlike the dehydrogenasecomplexes, these enzymes contain iron-sulfur clusters, utilize differentcofactors, and use ferredoxin or flavodixin as electron acceptors inlieu of NAD(P)H. While most enzymes in this family are specific topyruvate as a substrate (POR) some 2-keto-acid:ferredoxinoxidoreductases have been shown to accept a broad range of 2-ketoacidsas substrates including alpha-ketoglutarate and 2-oxobutanoate (Fukudaand Wakagi Biochim. Biophys. Acta 1597:74-80 (2002); Zhang et al. J.Biochem. 120:587-599 (1996)). One such enzyme is the OFOR from thethermoacidophilic archaeon Sulfolobus tokodaii 7, which contains analpha and beta subunit encoded by gene ST2300 (Fukuda and WakagiBiochim. Biophys. Acta 1597:74-80 (2002); Zhang et al. J. Biochem.120:587-599 (1996)). A plasmid-based expression system has beendeveloped for efficiently expressing this protein in E. coli (Fukuda etal. Eur. J. Biochem. 268:5639-5646 (2001)) and residues involved insubstrate specificity were determined (Fukuda and Wakagi Biochim.Biophys. Acta 1597:74-80 (2002)). Two OFORs from Aeropyrum pernix str.K1 have also been recently cloned into E. coli, characterized, and foundto react with a broad range of 2-oxoacids (Nishizawa et al. FEBS Lett.579:2319-2322 (2005)). The gene sequences of these OFOR candidates areavailable, although they do not have GenBank identifiers assigned todate. There is bioinformatic evidence that similar enzymes are presentin all archaea, some anaerobic bacteria and amitochondrial eukarya(Fukuda and Wakagi Biochim. Biophys. Acta 1597:74-80 (2005)). This classof enzyme is also interesting from an energetic standpoint, as reducedferredoxin could be used to generate NADH by ferredoxin-NAD reductase(Petitdemange et al. Biochim. Biophys. Acta 421:334-337 (1976)). Also,since most of the enzymes are designed to operate under anaerobicconditions, less enzyme engineering may be required relative to enzymesin the 2-keto-acid dehydrogenase complex family for activity in ananaerobic environment.

Gene Accession No. GI No. Organism ST2300 NP_378302.1 15922633Sulfolobus tokodaii 71.2.1.d Oxidoreductase (Phosphonate Reductase).

The conversion of 4-hydroxybutyryl-phosphate to 4-hydroxybutanal can becatalyzed by an oxidoreductase in the EC class 1.2.1. Aspartatesemialdehyde dehydrogenase (ASD, EC 1.2.1.11) catalyzes theNADPH-dependent reduction of 4-aspartyl phosphate toaspartate-4-semialdehyde. ASD participates in amino acid biosynthesisand recently has been studied as an antimicrobial target (Hadfield etal., Biochemistry 40:14475-14483 (2001). The E. coli ASD structure hasbeen solved (Hadfield et al., J. Mol. Biol. 289:991-1002 (1999)) and theenzyme has been shown to accept the alternate substratebeta-3-methylaspartyl phosphate (Shames et al., J. Biol. Chem.259:15331-15339 (1984)). The Haemophilus influenzae enzyme has been thesubject of enzyme engineering studies to alter substrate bindingaffinities at the active site (Blanco et al., Acta Crystallogr. D. Biol.Crystallogr. 60:1388-1395 (2004); Blanco et al., Acta Crystallogr. D.Biol. Crystallogr. 60:1808-1815 (2004)). Other ASD candidates are foundin Mycobacterium tuberculosis (Shafiani et al., J. Appl. Microbiol.98:832-838 (2005), Methanococcus jannaschii (Faehnle et al., J. Mol.Biol. 353:1055-1068 (2005)), and the infectious microorganisms Vibriocholera and Heliobacter pylori (Moore et al., Protein Expr. Pur25:189-194 (2002)). A related enzyme candidate isacetylglutamylphosphate reductase (EC 1.2.1.38), an enzyme thatnaturally reduces acetylglutamylphosphate toacetylglutamate-5-semialdehyde, found in S. cerevisiae (Pauwels et al.,Eur. J. Biochem. 270:1014-1024 (2003), B. subtilis (O'Reilly and Devine,Microbiology 140 (Pt 5):1023-1025 (1994)). and other organisms.

Gene Accession No. GI No. Organism asd NP_417891.1 16131307 Escherichiacoli asd YP_248335.1 68249223 Haemophilus influenzae asd AAB499961899206 Mycobacterium tuberculosis VC2036 NP_231670 15642038 Vibriocholera asd YP_002301787.1 210135348 Heliobacter pylori ARG5,6NP_010992.1 6320913 Saccharomyces cerevisiae argC NP_389001.1 16078184Bacillus subtilis

Other exemplary enzymes in this class include glyceraldehyde 3-phosphatedehydrogenase which converts glyceraldehyde-3-phosphate into D-glycerate1,3-bisphosphate (for example, E. coli gapA (Branlant and Branlant, Eur.J. Biochem. 150:61-66 (1985)), N-acetyl-gamma-glutamyl-phosphatereductase which converts N-acetyl-L-glutamate-5-semialdehyde intoN-acetyl-L-glutamyl-5-phosphate (for example, E. coli argC (Parsot etal., Gene 68:275-283 (1988)), and glutamate-5-semialdehydedehydrogenase, which converts L-glutamate-5-semialdehyde intoL-glutamyl-5-phospate (for example, E. coli proA (Smith et al., J.Bacteriol. 157:545-551 (1984)). Genes encoding glutamate-5-semialdehydedehydrogenase enzymes from Salmonella typhimurium (Mahan and Csonka, J.Bacteriol. 156:1249-1262 (1983)) and Campylobacter jejuni (Louie andChan, Mol. Gen. Genet. 240:29-35 (1993)) were cloned and expressed in E.coli.

Gene Accession No. GI No. Organism gapA P0A9B2.2 71159358 Escherichiacoli argC NP_418393.1 16131796 Escherichia coli proA NP_414778.116128229 Escherichia coli proA NP_459319.1 16763704 Salmonellatyphimurium proA P53000.2 9087222 Campylobacter jejuni1.2.1.e Acid Reductase.

Several steps in FIGS. 58, 62 and 63 depict the conversion ofunactivated acids to aldehydes by an acid reductase. These include theconversion of 4-hydroxybutyrate, succinate, alpha-ketoglutarate, and4-aminobutyrate to 4-hydroxybutanal, succinate semialdehyde,2,5-dioxopentanoate, and 4-aminobutanal, respectively. One notablecarboxylic acid reductase can be found in Nocardia iowensis whichcatalyzes the magnesium, ATP and NADPH-dependent reduction of carboxylicacids to their corresponding aldehydes (Venkitasubramanian et al., J.Biol. Chem. 282:478-485 (2007)). This enzyme is encoded by the car geneand was cloned and functionally expressed in E. coli (Venkitasubramanianet al., J. Biol. Chem. 282:478-485 (2007)). Expression of the npt geneproduct improved activity of the enzyme via post-transcriptionalmodification. The npt gene encodes a specific phosphopantetheinetransferase (PPTase) that converts the inactive apo-enzyme to the activeholo-enzyme. The natural substrate of this enzyme is vanillic acid, andthe enzyme exhibits broad acceptance of aromatic and aliphaticsubstrates (Venkitasubramanian et al., in Biocatalysis in thePharmaceutical and Biotechnology Industires, ed. R.N. Patel, Chapter 15,pp. 425-440, CRC Press LLC, Boca Raton, Fla. (2006)).

Gene Accession No. GI No. Organism car AAR91681.1 40796035 Nocardiaiowensis (sp. NRRL 5646) npt ABI83656.1 114848891 Nocardia iowensis (sp.NRRL 5646)

Additional car and npt genes can be identified based on sequencehomology.

Gene Accession No. GI No. Organism fadD9 YP_978699.1 121638475Mycobacterium bovis BCG BCG_2812c YP_978898.1 121638674 Mycobacteriumbovis BCG nfa20150 YP_118225.1 54023983 Nocardia farcinica IFM 10152nfa40540 YP_120266.1 54026024 Nocardia farcinica IFM 10152 SGR_6790YP_001828302.1 182440583 Streptomyces griseus subsp. griseus NBRC 13350SGR_665 YP_001822177.1 182434458 Streptomyces griseus subsp. griseusNBRC 13350

An additional enzyme candidate found in Streptomyces griseus is encodedby the griC and griD genes. This enzyme is believed to convert3-amino-4-hydroxybenzoic acid to 3-amino-4-hydroxybenzaldehyde asdeletion of either griC or griD led to accumulation of extracellular3-acetylamino-4-hydroxybenzoic acid, a shunt product of3-amino-4-hydroxybenzoic acid metabolism (Suzuki, et al., J. Antibiot.60(6):380-387 (2007)). Co-expression of griC and griD with SGR_665, anenzyme similar in sequence to the Nocardia iowensis npt, can bebeneficial.

Gene Accession No. GI No. Organism griC 182438036 YP_001825755.1Streptomyces griseus subsp. griseus NBRC 13350 griD 182438037YP_001825756.1 Streptomyces griseus subsp. griseus NBRC 13350 MSMEG_2956YP_887275.1 YP_887275.1 Mycobacterium smegmatis MC2 155 MSMEG_5739YP_889972.1 118469671 Mycobacterium smegmatis MC2 155 MSMEG_2648YP_886985.1 118471293 Mycobacterium smegmatis MC2 155 MAP1040cNP_959974.1 41407138 Mycobacterium avium subsp. paratuberculosis K-10MAP2899c NP_961833.1 41408997 Mycobacterium avium subsp.paratuberculosis K-10 MMAR_2117 YP_001850422.1 183982131 Mycobacteriummarinum M MMAR_2936 YP_001851230.1 183982939 Mycobacterium marinum MMMAR_1916 YP_001850220.1 183981929 Mycobacterium marinum MTpauDRAFT_33060 ZP_04027864.1 227980601 Tsukamurella paurometabola DSM20162 TpauDRAFT_20920 ZP_04026660.1 227979396 Tsukamurella paurometabolaDSM 20162 CPCC7001_1320 ZP_05045132.1 254431429 Cyanobium PCC7001DDBDRAFT_0187729 XP_636931.1 66806417 Dictyostelium discoideum AX4

An enzyme with similar characteristics, alpha-aminoadipate reductase(AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in somefungal species. This enzyme naturally reduces alpha-aminoadipate toalpha-aminoadipate semialdehyde. The carboxyl group is first activatedthrough the ATP-dependent formation of an adenylate that is then reducedby NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizesmagnesium and requires activation by a PPTase. Enzyme candidates for AARand its corresponding PPTase are found in Saccharomyces cerevisiae(Morris et al., Gene 98:141-145 (1991)), Candida albicans (Guo et al.,Mol. Genet. Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe(Ford et al., Curr. Genet. 28:131-137 (1995)). The AAR from S. pombeexhibited significant activity when expressed in E. coli (Guo et al.,Yeast 21:1279-1288 (2004)). The AAR from Penicillium chrysogenum acceptsS-carboxymethyl-L-cysteine as an alternate substrate, but did not reactwith adipate, L-glutamate or diaminopimelate (Hijarrubia et al., J.Biol. Chem. 278:8250-8256 (2003)). The gene encoding the P. chrysogenumPPTase has not been identified to date.

Gene Accession No. GI No. Organism LYS2 AAA34747.1 171867 Saccharomycescerevisiae LYS5 P50113.1 1708896 Saccharomyces cerevisiae LYS2AAC02241.1 2853226 Candida albicans LYS5 AAO26020.1 28136195 Candidaalbicans Lys1p P40976.3 13124791 Schizosaccharomyces pombe Lys7pQ10474.1 1723561 Schizosaccharomyces pombe Lys2 CAA74300.1 3282044Penicillium chrysogenum1.4.1.a Oxidoreductase (Aminating).

Glutamate dehydrogenase (Step J, FIGS. 62 and 63), 4-aminobutyratedehydrogenase (Step M, FIGS. 62 and 63), putrescine dehydrogenase (StepD, FIG. 63), 5-amino-2-oxopentanoate dehydrogenase (Step P, FIG. 63),and ornithine dehydrogenase (Step S, FIG. 63) can be catalyzed byaminating oxidoreductases. Enzymes in this EC class catalyze theoxidative deamination of alpha-amino acids with NAD+ or NADP+ asacceptor, and the reactions are typically reversible. Exemplaryoxidoreductases operating on amino acids include glutamate dehydrogenase(deaminating), encoded by gdhA, leucine dehydrogenase (deaminating),encoded by ldh, and aspartate dehydrogenase (deaminating), encoded bynadX. The gdhA gene product from Escherichia coli (Korber et al., J.Mol. Biol. 234:1270-1273 (1993); McPherson and Wootton, Nucleic AcidsRes. 11:5257-5266 (1983)), gdh from Thermotoga maritima (Kort et al.,Extremophiles 1:52-60 (1997); Lebbink, et al. J. Mol. Biol. 280:287-296(1998); Lebbink et al. J. Mol. Biol. 289:357-369 (1999)), and gdhA1 fromHalobacterium salinarum (Ingoldsby et al., Gene 349:237-244 (2005))catalyze the reversible interconversion of glutamate to 2-oxoglutarateand ammonia, while favoring NADP(H), NAD(H), or both, respectively. Theldh gene of Bacillus cereus encodes the LeuDH protein that has a wide ofrange of substrates including leucine, isoleucine, valine, and2-aminobutanoate (Ansorge and Kula, Biotechnol. Bioeng. 68:557-562(2000); Stoyan et al. J. Biotechnol. 54:77-80 (1997)). The nadX genefrom Thermotoga maritime encoding for the aspartate dehydrogenase isinvolved in the biosynthesis of NAD (Yang et al., J. Biol. Chem.278:8804-8808 (2003)).

Gene Accession No. GI No. Organism gdhA P00370 118547 Escherichia coligdh P96110.4 6226595 Thermotoga maritima gdhA1 NP_279651.1 15789827Halobacterium salinarum ldh P0A393 61222614 Bacillus cereus nadXNP_229443.1 15644391 Thermotoga maritima

Additional glutamate dehydrogenase gene candidates are found in Bacillussubtilis (Khan et al., Biosci. Biotechnol. Biochem. 69:1861-1870(2005)), Nicotiana tabacum (Purnell et al., Planta 222:167-180 (2005)),Oryza sativa (Abiko et al., Plant Cell Physiol. 46:1724-1734 (2005)),Haloferax mediterranei (Diaz et al., Extremophiles 10:105-115 (2006))and Halobactreium salinarum (Hayden et al., FEMS Microbiol. Lett.211:37-41 (2002)). The Nicotiana tabacum enzyme is composed of alpha andbeta subunits encoded by gdh1 and gdh2 (Purnell et al., Planta222:167-180 (2005)). Overexpression of the NADH-dependent glutamatedehydrogenase was found to improve ethanol production in engineeredstrains of S. cerevisiae (Roca et al., Appl. Environ. Microbiol.69:4732-4736 (2003)).

Gene Accession No. GI No. Organism rocG NP_391659.1 16080831 Bacillussubtilis gdh1 AAR11534.1 38146335 Nicotiana tabacum gdh2 AAR11535.138146337 Nicotiana tabacum GDH Q852M0 75243660 Oryza sativa GDH Q977U674499858 Haloferax mediterranei GDH P29051 118549 Halobactreiumsalinarum GDH2 NP_010066.1 6319986 Saccharomyces cerevisiae

An exemplary enzyme for catalyzing the conversion of aldehydes to theircorresponding primary amines is lysine 6-dehydrogenase (EC 1.4.1.18),encoded by the lysDH genes. The lysine 6-dehydrogenase (deaminating),encoded by lysDH gene, catalyze the oxidative deamination of the ε-aminogroup of L-lysine to form 2-aminoadipate-6-semialdehyde, which in turnnonenzymatically cyclizes to form Δ1-piperideine-6-carboxylate (Misonoand Nagasaki, J. Bacteriol. 150:398-401 (1982)). The lysDH gene fromGeobacillus stearothermophilus encodes a thermophilic NAD-dependentlysine 6-dehydrogenase (Heydari et al., Appl. Environ. Microbiol.70:937-942 (2004)). The lysDH gene from Aeropyrum pernix K1 isidentified through homology from genome projects. Additional enzymes canbe found in Agrobacterium tumefaciens (Hashimoto et al., J. Biochem.106:76-80 (1989); Misono and Nagasaki, J. Bacteriol. 150:398-401 (1982))and Achromobacter denitrificans (Ruldeekulthamrong et al., BMB. Rep.41:790-795 (2008)).

Gene Accession No. GI No. Organism lysDH BAB39707 13429872 Geobacillusstearothermophilus lysDH NP_147035.1 14602185 Aeropyrum pernix K1 lysDHNP_353966 15888285 Agrobacterium tumefaciens lysDH AAZ94428 74026644Achromobacter denitrificans

An enzyme that converts 3-oxoacids to 3-amino acids is3,5-diaminohexanoate dehydrogenase (EC 1.4.1.11), an enzyme found inorganisms that ferment lysine. The gene encoding this enzyme, kdd, wasrecently identified in Fusobacterium nucleatum (Kreimeyer et al., J.Biol. Chem. 282:7191-7197 (2007)). The enzyme has been purified andcharacterized in other organisms (Baker et al., J. Biol. Chem.247:7724-7734 (1972); Baker and van der Drift, Biochemistry 13:292-299(1974)), but the genes associated with these enzymes are not known.Candidates in Myxococcus xanthus, Porphyromonas gingivalis W83 and othersequenced organisms can be inferred by sequence homology.

Gene Accession No. GI No. Organism kdd AAL93966.1 19713113 Fusobacteriumnucleatum mxan_4391 ABF87267.1 108462082 Myxococcus xanthus pg_1069AAQ66183.1 34397119 Porphyromonas gingivalis2.3.1.a Acyltransferase (Transferring Phosphate Group to CoA).

Step P of FIG. 62 depicts the transformation of 4-hydroxybutyryl-CoA to4-hydroxybutyryl-Pi. Exemplary phosphate transferring acyltransferasesinclude phosphotransacetylase, encoded by pta, andphosphotransbutyrylase, encoded by ptb. The pta gene from E. coliencodes an enzyme that can convert acetyl-CoA into acetyl-phosphate, andvice versa (Suzuki, Biochim. Biophys. Acta 191:559-569 (1969)). Thisenzyme can also utilize propionyl-CoA instead of acetyl-CoA formingpropionate in the process (Hesslinger et al., Mol. Microbiol. 27:477-492(1998)). Similarly, the ptb gene from C. acetobutylicum encodes anenzyme that can convert butyryl-CoA into butyryl-phosphate (Walter etal., Gene 134:107-111 (1993)); Huang et al., J Mol. Microbiol.Biotechnol 2:33-38 (2000). Additional ptb genes can be found inbutyrate-producing bacterium L2-50 (Louis et al., J. Bacteriol.186:2099-2106 (2004)) and Bacillus megaterium (Vazquez et al., Curr.Microbiol. 42:345-349 (2001)).

Gene Accession No. GI No. Organism pta NP_416800.1 16130232 Escherichiacoli ptb NP_349676 15896327 Clostridium acetobutylicum ptb AAR19757.138425288 butyrate-producing bacterium L2-50 ptb CAC07932.1 10046659Bacillus megaterium2.6.1. Aminotransferase.

Aminotransferases reversibly convert an aldehyde or ketone to an aminogroup. Common amino donor/acceptor combinations includeglutamate/alpha-ketoglutarate, alanine/pyruvate, andaspartate/oxaloacetate. Several enzymes have been shown to convertaldehydes to primary amines, and vice versa, such as 4-aminobutyrate,putrescine, and 5-amino-2-oxopentanoate. These enzymes are particularlywell suited to carry out the following transformations: Step N in FIGS.62 and 63, Steps E and Q in FIG. 63. Lysine-6-aminotransferase (EC2.6.1.36) is one exemplary enzyme capable of forming a primary amine.This enzyme function, converting lysine to alpha-aminoadipatesemialdehyde, has been demonstrated in yeast and bacteria. Candidatesfrom Candida utilis (Hammer and Bode, J. Basic Microbiol. 32:21-27(1992)), Flavobacterium lutescens (Fujii et al., J. Biochem. 128:391-397(2000)) and Streptomyces clavuligenus (Romero et al., J. Ind. Microbiol.Biotechnol. 18:241-246 (1997)) have been characterized. A recombinantlysine-6-aminotransferase from S. clavuligenus was functionallyexpressed in E. coli (Tobin et al., J. Bacteriol. 173:6223-6229 (1991)).The F. lutescens enzyme is specific to alpha-ketoglutarate as the aminoacceptor (Soda and Misono, Biochemistry 7:4110-4119 (1968)). Otherenzymes which convert aldehydes to terminal amines include the dat geneproduct in Acinetobacter baumanii encoding2,4-diaminobutanoate:2-ketoglutarate 4-transaminase (Ikai and Yamamoto,J. Bacteriol. 179:5118-5125 (1997)). In addition to its naturalsubstrate, 2,4-diaminobutyrate, DAT transaminates the terminal amines oflysine, 4-aminobutyrate and ornithine.

Gene Accession No. GI No. Organism lat BAB13756.1 10336502Flavobacterium lutescens lat AAA26777.1 153343 Streptomyces clavuligenusdat P56744.1 6685373 Acinetobacter baumanii

The conversion of an aldehyde to a terminal amine can also be catalyzedby gamma-aminobutyrate transaminase (GABA transaminase or4-aminobutyrate transaminase). This enzyme naturally interconvertssuccinic semialdehyde and glutamate to 4-aminobutyrate andalpha-ketoglutarate and is known to have a broad substrate range (Liu etal., Biochemistry 43:10896-10905 2004); Schulz et al., Appl. Environ.Microbiol. 56:1-6 (1990)). The two GABA transaminases in E. coli areencoded by gabT (Bartsch et al., J. Bacteriol. 172:7035-7042 (1990)) andpuuE (Kurihara et al., J. Biol. Chem. 280:4602-4608. (2005)). GABAtransaminases in Mus musculus, Pseudomonas fluorescens, and Sus scrofahave been shown to react with a range of alternate substrates including6-aminocaproic acid (Cooper, Methods Enzymol. 113:80-82 (1985); Scottand Jakoby, J. Biol. Chem. 234:932-936 (1959)).

Gene Accession No. GI No. Organism gabT NP_417148.1 16130576 Escherichiacoli puuE NP_415818.1 16129263 Escherichia coli abat NP_766549.237202121 Mus musculus gabT YP_257332.1 70733692 Pseudomonas fluorescensabat NP_999428.1 47523600 Sus scrofa

Additional enzyme candidates for interconverting aldehydes and primaryamines are putrescine transminases or other diamine aminotransferases.The E. coli putrescine aminotransferase is encoded by the ygjG gene, andthe purified enzyme also was able to transaminate cadaverine andspermidine (Samsonova et al., BMC Microbiol. 3:2 (2003)). In addition,activity of this enzyme on 1,7-diaminoheptane and with amino acceptorsother than 2-oxoglutarate (for example, pyruvate, 2-oxobutanoate) hasbeen reported (Kim, J. Biol. Chem. 239:783-786 (1964); Samsonova et al.,BMC Microbiol. 3:2 (2003)). A putrescine aminotransferase with higheractivity with pyruvate as the amino acceptor than alpha-ketoglutarate isthe spuC gene of Pseudomonas aeruginosa (Lu et al., J. Bacteriol.184:3765-3773 (2002)).

Gene Accession No. GI No. Organism ygjG NP_417544 145698310 Escherichiacoli spuC AAG03688 9946143 Pseudomonas aeruginosa

Enzymes that transaminate 3-oxoacids include GABA aminotransferase(described above), beta-alanine/alpha-ketoglutarate aminotransferase and3-amino-2-methylpropionate aminotransferase.Beta-alanine/alpha-ketoglutarate aminotransferase (WO08027742) reactswith beta-alanine to form malonic semialdehyde, a 3-oxoacid. The geneproduct of SkPYD4 in Saccharomyces kluyveri was shown to preferentiallyuse beta-alanine as the amino group donor (Andersen and Hansen, Gene124:105-109 (1993)). SkUGA1 encodes a homologue of Saccharomycescerevisiae GABA aminotransferase, UGA1 (Ramos et al., Eur. J. Biochem.149:401-404 (1985)), whereas SkPYD4 encodes an enzyme involved in bothbeta-alanine and GABA transamination (Andersen and Hansen, Gene124:105-109 (1993)). 3-Amino-2-methylpropionate transaminase catalyzesthe transformation from methylmalonate semialdehyde to3-amino-2-methylpropionate. The enzyme has been characterized in Rattusnorvegicus and Sus scrofa and is encoded by Abat (Kakimoto et al.,Biochim. Biophys. Acta 156:374-380 (1968); Tamaki et al., MethodsEnzymol. 324:376-389 (2000)).

Gene Accession No. GI No. Organism SkyPYD4 ABF58893.1 98626772 Lachanceakluyveri SkUGA1 ABF58894.1 98626792 Lachancea kluyveri UGA1 NP_011533.16321456 Saccharomyces cerevisiae Abat P50554.3 122065191 Rattusnorvegicus Abat P80147.2 120968 Sus scrofa

Several aminotransferases transaminate the amino groups of amino acidsto form 2-oxoacids. Aspartate aminotransferase is an enzyme thatnaturally transfers an oxo group from oxaloacetate to glutamate, formingalpha-ketoglutarate and aspartate. Aspartate is similar in structure toOHED and 2-AHD. Aspartate aminotransferase activity is catalyzed by, forexample, the gene products of aspC from Escherichia coli (Yagi et al.,FEBS Lett. 100:81-84 (1979); Yagi et al., Methods Enzymol. 113:83-89(1985)), AAT2 from Saccharomyces cerevisiae (Yagi et al., J. Biochem.92:35-43 (1982)) and ASPS from Arabidopsis thaliana (de la Torre et al.,Plant J. 46:414-425 (2006); Kwok and Hanson. J. Exp. Bot. 55:595-604(2004); Wilkie and Warren, Protein Expr. Pur 12:381-389 (1998)). Theenzyme from Rattus norvegicus has been shown to transaminate alternatesubstrates such as 2-aminohexanedioic acid and 2,4-diaminobutyric acid(Recasens et al., Biochemistry 19:4583-4589 (1980)). Aminotransferasesthat work on other amino-acid substrates can also be able to catalyzethis transformation. Valine aminotransferase catalyzes the conversion ofvaline and pyruvate to 2-ketoisovalerate and alanine The E. coli gene,avtA, encodes one such enzyme (Whalen and Berg, J. Bacteriol.150:739-746 (1982)). This gene product also catalyzes the transaminationof α-ketobutyrate to generate α-aminobutyrate, although the amine donorin this reaction has not been identified (Whalen and Berg, J. Bacteriol.158:571-574 1984)). The gene product of the E. coli serC catalyzes tworeactions, phosphoserine aminotransferase and phosphohydroxythreonineaminotransferase (Lam and Winkler, J. Bacteriol. 172:6518-6528 (1990)),and activity on non-phosphorylated substrates could not be detected(Drewke et al., FEBS Lett. 390:179-182 (1996)).

Gene Accession No. GI No. Organism aspC NP_415448.1 16128895 Escherichiacoli AAT2 P23542.3 1703040 Saccharomyces cerevisiae ASP5 P46248.220532373 Arabidopsis thaliana Got2 P00507 112987 Rattus norvegicus avtAYP_026231.1 49176374 Escherichia coli serC NP_415427.1 16128874Escherichia coli

Another enzyme candidate is alpha-aminoadipate aminotransferase (EC2.6.1.39), an enzyme that participates in lysine biosynthesis anddegradation in some organisms. This enzyme interconverts 2-aminoadipateand 2-oxoadipate, using alpha-ketoglutarate as the amino acceptor. Genecandidates are found in Homo sapiens (Okuno et al., Enzyme Protein47:136-148 (1993)) and Thermus thermophilus (Miyazaki et al.,Microbiology 150:2327-2334 (2004)). The Thermus thermophilus enzyme,encoded by lysN, is active with several alternate substrates includingoxaloacetate, 2-oxoisocaproate, 2-oxoisovalerate, and2-oxo-3-methylvalerate.

Gene Accession No. GI No. Organism lysN BAC76939.1 31096548 Thermusthermophilus AadAT-II Q8N5Z0.2 46395904 Homo sapiens2.7.2.a Phosphotransferase (Carboxy Acceptor).

Phosphotransferase enzymes in the EC class 2.7.2 transform carboxylicacids to phosphonic acids with concurrent hydrolysis of one ATP. Step 0of FIG. 62 involves the conversion of 4-hydroxybutyrate to4-hydroxybutyryl-phosphate by such an enzyme. Butyrate kinase (EC2.7.2.7) carries out the reversible conversion of butyryl-phosphate tobutyrate during acidogenesis in C. acetobutylicum (Cary et al., Appl.Environ. Microbiol. 56:1576-1583 (1990)). This enzyme is encoded byeither of the two buk gene products (Huang et al., J. Mol. Microbiol.Biotechnol. 2:33-38 (2000)). Other butyrate kinase enzymes are found inC. butyricum and C. tetanomorphum (Twarog and Wolfe, J. Bacteriol.86:112-117 (1963)). Related enzyme isobutyrate kinase from Thermotogamaritima has also been expressed in E. coli and crystallized (Diao etal., Acta Crystallogr. D. Biol. Crystallogr. 59:1100-1102 (2003); Diaoand Hasson, J. Bacteriol. 191:2521-2529 (2009)). Aspartokinase catalyzesthe ATP-dependent phosphorylation of aspartate and participates in thesynthesis of several amino acids. The aspartokinase III enzyme in E.coli, encoded by lysC, has a broad substrate range, and the catalyticresidues involved in substrate specificity have been elucidated (Kengand Viola, Arch. Biochem. Biophys. 335:73-81 (1996)). Two additionalkinases in E. coli are also good candidates: acetate kinase andgamma-glutamyl kinase. The E. coli acetate kinase, encoded by ackA(Skarstedt and Silverstein, J. Biol. Chem. 251:6775-6783 (1976)),phosphorylates propionate in addition to acetate (Hesslinger et al.,Mol. Microbiol. 27:477-492 (1998)). The E. coli gamma-glutamyl kinase,encoded by proB (Smith et al., J. Bacteriol. 157:545-551 (1984)),phosphorylates the gamma carbonic acid group of glutamate.

Gene Accession No. GI No. Organism buk1 NP_349675 15896326 Clostridiumacetobutylicum buk2 Q97II1 20137415 Clostridium acetobutylicum buk2Q9X278.1 6685256 Thermotoga maritima lysC NP_418448.1 16131850Escherichia coli ackA NP_416799.1 16130231 Escherichia coli proBNP_414777.1 16128228 Escherichia coli

Acetylglutamate kinase phosphorylates acetylated glutamate duringarginine biosynthesis. This enzyme is not known to accept alternatesubstrates; however, several residues of the E. coli enzyme involved insubstrate binding and phosphorylation have been elucidated bysite-directed mutagenesis (Marco-Marin et al., J. Mol. Biol. 334:459-476(2003); Ramon-Maiques et al., Structure 10:329-342 (2002)). The enzymeis encoded by argB in Bacillus subtilis and E. coli (Parsot et al., Gene68:275-283 (1988)), and ARG5,6 in S. cerevisiae (Pauwels et al., Eur. J.Biochem. 270:1014-1024 (2003)). The ARG5,6 gene of S. cerevisiae encodesa polyprotein precursor that is matured in the mitochondrial matrix tobecome acetylglutamate kinase and acetylglutamylphosphate reductase.

Gene Accession No. GI No. Organism argB NP_418394.3 145698337Escherichia coli argB NP_389003.1 16078186 Bacillus subtilis ARG5,6NP_010992.1 6320913 Saccharomyces cerevisiae2.8.3.a CoA Transferase.

The gene products of cat1, cat2, and cat3 of Clostridium kluyveri havebeen shown to exhibit succinyl-CoA (Step G, FIGS. 62 and 63),4-hydroxybutyryl-CoA (Step T, FIG. 62), and butyryl-CoAacetyltransferase activity, respectively (Seedorf et al., Proc. Natl.Acad. Sci. USA 105:2128-2133 (2008); Sohling and Gottschalk, J Bacteriol178:871-880 (1996)). Similar CoA transferase activities are also presentin Trichomonas vaginalis (van Grinsven et al., J. Biol. Chem.283:1411-1418 (2008)) and Trypanosoma brucei (Riviere et al., J. Biol.Chem. 279:45337-45346 (2004)).

Gene Accession No. GI No. Organism cat1 P38946.1 729048 Clostridiumkluyveri cat2 P38942.2 1705614 Clostridium kluyveri cat3 EDK35586.1146349050 Clostridium kluyveri TVAG_395550 XP_001330176 123975034Trichomonas vaginalis G3 Tb11.02.0290 XP_828352 71754875 Trypanosomabrucei

An additionally useful enzyme for this type of transformation isacyl-CoA:acetate-CoA transferase, also known as acetate-CoA transferase(EC 2.8.3.8), which has been shown to transfer the CoA moiety to acetatefrom a variety of branched and linear acyl-CoA substrates, includingisobutyrate (Matthies and Schink, Appl. Environ. Microbiol. 58:1435-1439(1992)), valerate (Vanderwinkel et al., Biochem. Biophys. Res. Commun.33:902-908 (1968)) and butanoate (Vanderwinkel, supra (1968)). Thisenzyme is encoded by atoA (alpha subunit) and atoD (beta subunit) in E.coli sp. K12 (Korolev et al., Acta Crystallogr. D Biol. Crystallogr.58:2116-2121 (2002); Vanderwinkel, supra (1968)). Similar enzymes existin Corynebacterium glutamicum ATCC 13032 (Duncan et al., Appl. Environ.Microbiol. 68:5186-5190 (2002)), Clostridium acetobutylicum (Cary etal., Appl. Environ. Microbiol. 56:1576-1583 (1990); Wiesenborn et al.,Appl. Environ. Microbiol. 55:323-329 (1989)), and Clostridiumsaccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol. Biochem.71:58-68 (2007)).

Gene Accession No. GI No. Organism atoA P76459.1 2492994 Escherichiacoli K12 atoD P76458.1 2492990 Escherichia coli K12 actA YP_226809.162391407 Corynebacterium glutamicum cg0592 YP_224801.1 62389399Corynebacterium glutamicum ctfA NP_149326.1 15004866 Clostridiumacetobutylicum ctfB NP_149327.1 15004867 Clostridium acetobutylicum ctfAAAP42564.1 31075384 Clostridium saccharoperbutylacetonicum ctfBAAP42565.1 31075385 Clostridium saccharoperbutylacetonicum

The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from anaerobicbacterium Acidaminococcus fermentans reacts with diacid glutaconyl-CoAand 3-butenoyl-CoA (Mack and Buckel, FEBS Lett. 405:209-212 (1997)). Thegenes encoding this enzyme are gctA and gctB. This enzyme has reducedbut detectable activity with other CoA derivatives includingglutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckelet al., Eur. J. Biochem. 118:315-321 (1981)). The enzyme has been clonedand expressed in E. coli (Mac et al., Eur. J. Biochem. 226:41-51(1994)).

Gene Accession No. GI No. Organism gctA CAA57199.1 559392Acidaminococcus fermentans gctB CAA57200.1 559393 Acidaminococcusfermentans3.1.2.a CoA Hydrolase.

Enzymes in the 3.1.2 family hydrolyze acyl-CoA molecules to theircorresponding acids. However, such enzymes can be modified to empartCoA-ligase or synthetase functionality if coupled to an energy sourcesuch as a proton pump or direct ATP hydrolysis. Several eukaryoticacetyl-CoA hydrolases (EC 3.1.2.1) have broad substrate specificity. Forexample, the enzyme from Rattus norvegicus brain (Robinson et al.,Biochem. Biophys. Res. Commun. 71:959-965 (1976)) can react withbutyryl-CoA, hexanoyl-CoA and malonyl-CoA. Though its sequence has notbeen reported, the enzyme from the mitochondrion of the pea leaf alsohas a broad substrate specificity, with demonstrated activity onacetyl-CoA, propionyl-CoA, butyryl-CoA, palmitoyl-CoA, oleoyl-CoA,succinyl-CoA, and crotonyl-CoA (Zeiher and Randall, Plant. Physiol.94:20-27 (1990)). The acetyl-CoA hydrolase, ACH1, from S. cerevisiaerepresents another candidate hydrolase (Buu et al., J. Biol. Chem.278:17203-17209 (2003)).

Gene Accession No. GI No. Organism acot12 NP_570103.1 18543355 Rattusnorvegicus ACH1 NP_009538 6319456 Saccharomyces cerevisiae

Another candidate hydrolase is the human dicarboxylic acid thioesterase,acot8, which exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA,sebacyl-CoA, and dodecanedioyl-CoA (Westin et al., J. Biol. Chem.280:38125-38132 (2005)) and the closest E. coli homolog, tesB, which canalso hydrolyze a broad range of CoA thioesters (Naggert et al., J. Biol.Chem. 266:11044-11050 (1991)). A similar enzyme has also beencharacterized in the rat liver (Deana, Biochem. Int. 26:767-773 (1992)).Other potential E. coli thioester hydrolases include the gene productsof tesA (Bonner and Bloch, J. Biol. Chem. 247:3123-3133 (1972)), ybgC(Kuznetsova et al., FEMS Microbiol. Rev. 29:263-279 (2005); Zhuang etal., FEBS Lett. 516:161-163 (2002)), paaI (Song et al., J. Biol. Chem.281:11028-11038 (2006)), and ybdB (Leduc et al., J. Bacteriol.189:7112-7126 (2007)).

Gene Accession No. GI No. Organism acot8 CAA15502 3191970 Homo sapienstesB NP_414986 16128437 Escherichia coli acot8 NP_570112 51036669 Rattusnorvegicus tesA NP_415027 16128478 Escherichia coli ybgC NP_41526416128711 Escherichia coli paaI NP_415914 16129357 Escherichia coli ybdBNP_415129 16128580 Escherichia coli

Yet another candidate hydrolase is the glutaconate CoA-transferase fromAcidaminococcus fermentans. This enzyme was transformed by site-directedmutagenesis into an acyl-CoA hydrolase with activity on glutaryl-CoA,acetyl-CoA and 3-butenoyl-CoA (Mack and Buckel, FEBS Lett. 405:209-212(1997)). This indicates that the enzymes encodingsuccinyl-CoA:3-ketoacid-CoA transferases and acetoacetyl-CoA:acetyl-CoAtransferases can also serve as candidates for this reaction step butwould likely require certain mutations to change their function.

Gene Accession No. GI No. Organism gctA CAA57199.1 559392Acidaminococcus fermentans gctB CAA57200.1 559393 Acidaminococcusfermentans

Additional hydrolase enzymes include 3-hydroxyisobutyryl-CoA hydrolasewhich has been described to efficiently catalyze the conversion of3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate during valinedegradation (Shimomura et al., J. Biol. Chem. 269:14248-14253 (1994)).Genes encoding this enzyme include hibch of Rattus norvegicus (Shimomuraet al., supra (1994); Shimomura et al., Methods Enzymol. 324:229-240(2000)) and Homo sapiens (Shimomura et al., supra (1994). Candidategenes by sequence homology include hibch of Saccharomyces cerevisiae andBC_2292 of Bacillus cereus.

Gene Accession No. GI No. Organism hibch Q5XIE6.2 146324906 Rattusnorvegicus hibch Q6NVY1.2 146324905 Homo sapiens hibch P28817.2 2506374Saccharomyces cerevisiae BC_2292 AP09256 29895975 Bacillus cereus4.1.1.a Carboxy-Lyase.

Decarboxylation of Alpha-Keto Acids. Alpha-ketoglutarate decarboxylase(Step B, FIGS. 58, 62 and 63), 5-hydroxy-2-oxopentanoic aciddecarboxylase (Step Z, FIG. 62), and 5-amino-2-oxopentanoatedecarboxylase (Step R, FIG. 63) all involve the decarboxylation of analpha-ketoacid. The decarboxylation of keto-acids is catalyzed by avariety of enzymes with varied substrate specificities, includingpyruvate decarboxylase (EC 4.1.1.1), benzoylformate decarboxylase (EC4.1.1.7), alpha-ketoglutarate decarboxylase and branched-chainalpha-ketoacid decarboxylase.

Pyruvate decarboxylase (PDC), also termed keto-acid decarboxylase, is akey enzyme in alcoholic fermentation, catalyzing the decarboxylation ofpyruvate to acetaldehyde. The enzyme from Saccharomyces cerevisiae has abroad substrate range for aliphatic 2-keto acids including2-ketobutyrate, 2-ketovalerate, 3-hydroxypyruvate and 2-phenylpyruvate(Davie et al., J. Biol. Chem. 267:16601-16606 (1992)). This enzyme hasbeen extensively studied, engineered for altered activity, andfunctionally expressed in E. coli (Killenberg-Jabs et al., Eur. J.Biochem. 268:1698-1704 (2001); Li and Jordan, Biochemistry38:10004-10012 (1999); ter Schure et al., Appl. Environ. Microbiol.64:1303-1307 (1998)). The PDC from Zymomonas mobilus, encoded by pdc,also has a broad substrate range and has been a subject of directedengineering studies to alter the affinity for different substrates(Siegert et al., Protein Eng. Des. Sel. 18:345-357 (2005)). The crystalstructure of this enzyme is available (Killenberg-Jabs et al., Eur. J.Biochem. 268:1698-1704 (2001)). Other well-characterized PDC candidatesinclude the enzymes from Acetobacter pasteurians (Chandra et al., Arch.Microbiol. 176:443-451 (2001)) and Kluyveromyces lactis (Krieger et al.,Eur. J. Biochem. 269:3256-3263 (2002)).

Gene Accession No. GI No. Organism pdc P06672.1 118391 Zymomonas mobiluspdc1 P06169 30923172 Saccharomyces cerevisiae pdc AM21208 20385191Acetobacter pasteurians pdc1 Q12629 52788279 Kluyveromyces lactis

Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broadsubstrate range and has been the target of enzyme engineering studies.The enzyme from Pseudomonas putida has been extensively studied andcrystal structures of this enzyme are available (Hasson et al.,Biochemistry 37:9918-9930 (1998); Polovnikova et al., Biochemistry42:1820-1830 (2003). Site-directed mutagenesis of two residues in theactive site of the Pseudomonas putida enzyme altered the affinity (Km)of naturally and non-naturally occurring substrates (Siegert et al.,Protein Eng. Des. Sel. 18:345-357 (2005)). The properties of this enzymehave been further modified by directed engineering (Lingen et al.,Protein Eng. 15:585-593 (2002); Lingen et al., Chembiochem. 4:721-726(2003)). The enzyme from Pseudomonas aeruginosa, encoded by mdlC, hasalso been characterized experimentally (Barrowman et al., FEMSMicrobiol. Lett. 34:57-60 (1986)). Additional gene candidates fromPseudomonas stutzeri, Pseudomonas fluorescens and other organisms can beinferred by sequence homology or identified using a growth selectionsystem developed in Pseudomonas putida (Henning et al., Appl. Environ.Microbiol. 72:7510-7517 (2006)).

Gene Accession No. GI No. Organism mdlC P20906.2 3915757 Pseudomonasputida mdlC Q9HUR2.1 81539678 Pseudomonas aeruginosa dpgB ABN80423.1126202187 Pseudomonas stutzeri ilvB-1 YP_260581.1 70730840 Pseudomonasfluorescens

A third enzyme capable of decarboxylating 2-oxoacids isalpha-ketoglutarate decarboxylase (KGD). The substrate range of thisclass of enzymes has not been studied to date. The KDC fromMycobacterium tuberculosis (Tian et al., Proc. Natl. Acad. Sci. USA102:10670-10675 (2005)) has been cloned and functionally expressed.However, it is not an ideal candidate for strain engineering because itis large (˜130 kD) and GC-rich. KDC enzyme activity has been detected inseveral species of rhizobia including Bradyrhizobium japonicum andMesorhizobium loti (Green et al., J. Bacteriol. 182:2838-2844 (2000).Although the KDC-encoding gene(s) have not been isolated in theseorganisms, the genome sequences are available, and several genes in eachgenome are annotated as putative KDCs. A KDC from Euglena gracilis hasalso been characterized, but the gene associated with this activity hasnot been identified to date (Shigeoka and Nakano, Arch. Biochem.Biophys. 288:22-28 (1991)). The first twenty amino acids starting fromthe N-terminus were sequenced (MTYKAPVKDVKFLLDKVFKV; SEQ ID NO:45)(Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). Thegene can be identified by testing candidate genes containing thisN-terminal sequence for KDC activity.

Gene Accession No. GI No. Organism kgd O50463.4 160395583 Mycobacteriumtuberculosis kgd NP_767092.1 27375563 Bradyrhizobium japonicum kgdNP_105204.1 13473636 Mesorhizobium loti

A fourth candidate enzyme for catalyzing this reaction is branched chainalpha-ketoacid decarboxylase (BCKA). This class of enzyme has been shownto act on a variety of compounds varying in chain length from 3 to 6carbons (Oku and Kaneda, J. Biol. Chem. 263:18386-18396 (1988); Smit etal., B. A., J. E. Hylckama Vlieg, W. J. Engels, L. Meijer, J. T.Wouters, and G. Smit. Identification, cloning, and characterization of aLactococcus lactis branched-chain alpha-keto acid decarboxylase involvedin flavor formation. Appl. Environ. Microbiol. 71:303-311 (2005)). Theenzyme in Lactococcus lactis has been characterized on a variety ofbranched and linear substrates including 2-oxobutanoate, 2-oxohexanoate,2-oxopentanoate, 3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate andisocaproate (Smit et al., Appl. Environ. Microbiol. 71:303-311 (2005)).The enzyme has been structurally characterized (Berg et al., Science318:1782-1786 (2007)). Sequence alignments between the Lactococcusenzyme and the pyruvate decarboxylase of Zymomonas mobilus indicate thatthe catalytic and substrate recognition residues are nearly identical(Siegert et al., Protein Eng. Des. Sel. 18:345-357 (2005)), so thisenzyme would be a promising candidate for directed engineering.Decarboxylation of alpha-ketoglutarate by a BCKA was detected inBacillus subtilis; however, this activity was low (5%) relative toactivity on other branched-chain substrates (Oku and Kaneda.Biosynthesis of branched-chain fatty acids in Bacillus subtilis. Adecarboxylase is essential for branched-chain fatty acid synthetase. J.Biol. Chem. 263:18386-18396 (1988)), and the gene encoding this enzymehas not been identified to date. Additional BCKA gene candidates can beidentified by homology to the Lactococcus lactis protein sequence. Manyof the high-scoring BLASTp hits to this enzyme are annotated asindolepyruvate decarboxylases (EC 4.1.1.74). Indolepyruvatedecarboxylase (IPDA) is an enzyme that catalyzes the decarboxylation ofindolepyruvate to indoleacetaldehyde in plants and plant bacteria.

Gene Accession No. GI No. Organism kdcA AAS49166.1 44921617 Lactococcuslactis

Recombinant branched chain alpha-keto acid decarboxylase enzymes derivedfrom the E1 subunits of the mitochondrial branched-chain keto aciddehydrogenase complex from Homo sapiens and Bos taurus have been clonedand functionally expressed in E. coli (Davie et al., J. Biol. Chem.267:16601-16606 1992); Wynn et al., J. Biol. Chem. 267:1881-1887 (1992);Wynn et al., J. Biol. Chem. 267:12400-12403 (1992)). In these studies,the authors found that co-expression of chaperonins GroEL and GroESenhanced the specific activity of the decarboxylase by 500-fold (Wynn etal., J. Biol. Chem. 267:12400-12403 (1992)). These enzymes are composedof two alpha and two beta subunits.

Gene Accession No. GI No. Organism BCKDHB NP_898871.1 34101272 Homosapiens BCKDHA NP_000700.1 11386135 Homo sapiens BCKDHB P21839 115502434Bos taurus BCKDHA P11178 129030 Bos taurus

Decarboxylation of Alpha-Keto Acids. Several ornithine decarboxylase(Step U, FIG. 63) enzymes also exhibit activity on lysine and othersimilar compounds. Such enzymes are found in Nicotiana glutinosa (Leeand Cho, Biochem. J. 360:657-665 (2001)), Lactobacillus sp. 30a (Guirardand Snell, J. Biol. Chem. 255:5960-5964 (1980)) and Vibrio vulnificus(Lee et al., J. Biol. Chem. 282:27115-27125 (2007)). The enzymes fromLactobacillus sp. 30a (Momany et al., J. Mol. Biol. 252:643-655 (1995))and V. vulnificus have been crystallized. The V. vulnificus enzymeefficiently catalyzes lysine decarboxylation, and the residues involvedin substrate specificity have been elucidated (Lee et al., J. Biol.Chem. 282:27115-27125 (2007)). A similar enzyme has been characterizedin Trichomonas vaginalis, but the gene encoding this enzyme is not known(Yarlett et al., Biochem. J. 293 (Pt 2):487-493 (1993)).

Gene Accession No. GI No. Organism AF323910.1: AAG45222.1 12007488Nicotiana glutinosa 1 . . . 1299 odc1 P43099.2 1169251 Lactobacillus sp.30a VV2_1235 NP_763142.1 27367615 Vibrio vulnificus

Glutamate decarboxylase enzymes (Step L, FIGS. 62 and 63) are alsowell-characterized. Exemplary glutamate decarboxylases can be found inE. coli (De Biase et al., Protein Expr. Pur 8:430-438 (1996)), S.cerevisiae (Coleman et al., J. Biol. Chem. 276:244-250 (2001)), and Homosapiens (Bu et al., Proc. Natl. Acad. Sci. USA 89:2115-2119 (1992); Buand Tobin, Genomics 21:222-228 (1994)).

Gene Accession No. GI No. Organism GAD1 NP_000808 58331246 Homo sapiensGAD2 NP_001127838 197276620 Homo sapiens gadA NP_417974 16131389Escherichia coli gadB NP_416010 16129452 Escherichia coli GAD1 NP_0139766323905 Saccharomyces cerevisiae

Lysine decarboxylase (EC 4.1.1.18) catalyzes the decarboxylation oflysine to cadaverine. Two isozymes of this enzyme are encoded in the E.coli genome by genes cadA and ldcC. CadA is involved in acid resistanceand is subject to positive regulation by the cadC gene product(Lemonnier and Lane, Microbiology 144 (Pt 3):751-760 (1998)). CadCaccepts hydroxylysine and S-aminoethylcysteine as alternate substrates,and 2-Aminopimelate and 6-ACA act as competitive inhibitors to thisenzyme (Sabo et al., Biochemistry 13:662-670 (1974)). Directed evolutionor other enzyme engineering methods can be utilized to increase theactivity for this enzyme to decarboxylate 2-aminopimelate. Theconstitutively expressed ldc gene product is less active than CadA(Lemonnier and Lane, Microbiology 144 (Pt 3):751-760 (1998)). A lysinedecarboxylase analogous to CadA was recently identified in Vibrioparahaemolyticus (Tanaka et al., J. Appl. Microbiol. 104:1283-1293(2008)). The lysine decarboxylase from Selenomonas ruminantium, encodedby ldc, bears sequence similarity to eukaryotic ornithinedecarboxylases, and accepts both L-lysine and L-ornithine as substrates(Takatsuka et al., Biosci. Biotechnol. Biochem. 63:1843-1846 (1999)).Active site residues were identified and engineered to alter thesubstrate specificity of the enzyme (Takatsuka et al., J. Bacteriol.182:6732-6741 (2000)).

Gene Accession No. GI No. Organism cadA AAA23536.1 145458 Escherichiacoli ldcC AAC73297.1 1786384 Escherichia coli ldc O50657.1 13124043Selenomonas ruminantium cadA AB124819.1 44886078 Vibrio parahaemolyticus6.2.1.a CoA Synthetase.

CoA synthetase or ligase reactions are required by Step I of FIGS. 62and 63, and Step V of FIG. 62. Succinate or 4-hydroxybutyrate are therequired substrates. Exemplary genes encoding enzymes likely to carryout these transformations include the sucCD genes of E. coli, whichnaturally form a succinyl-CoA synthetase complex. This enzyme complexnaturally catalyzes the formation of succinyl-CoA from succinate withthe concomitant consumption of one ATP, a reaction which is reversiblein vivo (Buck et al., Biochem. 24:6245-6252 (1985)).

Gene Accession No. GI No. Organism sucC NP_415256.1 16128703 Escherichiacoli sucD AAC73823.1 1786949 Escherichia coli

Additional exemplary CoA-ligases include the rat dicarboxylate-CoAligase for which the sequence is yet uncharacterized (Vamecq et al.,Biochemical J. 230:683-693 (1985)), either of the two characterizedphenylacetate-CoA ligases from P. chrysogenum (Lamas-Maceiras et al.,Biochem. J. 395:147-155 (2005); Wang et al., Biochem Biophy Res Commun360(2):453-458 (2007)), the phenylacetate-CoA ligase from Pseudomonasputida (Martinez-Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)),and the 6-carboxyhexanoate-CoA ligase from Bacillus subtilis (Bower etal., J. Bacteriol. 178(14):4122-4130 (1996)). Additional candidateenzymes are acetoacetyl-CoA synthetases from Mus musculus (Hasegawa etal., Biochim. Biophys. Acta 1779:414-419 (2008)) and Homo sapiens(Ohgami et al., Biochem. Pharmacol. 65:989-994 (2003)), which naturallycatalyze the ATP-dependant conversion of acetoacetate intoacetoacetyl-CoA. 4-Hydroxybutyryl-CoA synthetase activity has beendemonstrated in Metallosphaera sedula (Berg et al., Science318:1782-1786 (2007)). This function has been tentatively assigned tothe Msed_1422 gene.

Gene Accession No. GI No. Organism phl CAJ15517.1 77019264 Penicilliumchrysogenum phlB ABS19624.1 152002983 Penicillium chrysogenum paaFAAC24333.2 22711873 Pseudomonas putida bioW NP_390902.2 50812281Bacillus subtilis AACS NP_084486.1 21313520 Mus musculus AACSNP_076417.2 31982927 Homo sapiens Msed_1422 YP_001191504 146304188Metallosphaera sedula

ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is anothercandidate enzyme that couples the conversion of acyl-CoA esters to theircorresponding acids with the concurrent synthesis of ATP. Severalenzymes with broad substrate specificities have been described in theliterature. ACD I from Archaeoglobus fulgidus, encoded by AF1211, wasshown to operate on a variety of linear and branched-chain substratesincluding acetyl-CoA, propionyl-CoA, butyryl-CoA, acetate, propionate,butyrate, isobutyryate, isovalerate, succinate, fumarate, phenylacetate,indoleacetate (Musfeldt et al., J. Bacteriol. 184:636-644 (2002)). Theenzyme from Haloarcula marismortui (annotated as a succinyl-CoAsynthetase) accepts propionate, butyrate, and branched-chain acids(isovalerate and isobutyrate) as substrates, and was shown to operate inthe forward and reverse directions (Brasen et al., Arch. Microbiol.182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophiliccrenarchaeon Pyrobaculum aerophilum showed the broadest substrate rangeof all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA(preferred substrate) and phenylacetyl-CoA (Brasen et al., supra(2004)). The enzymes from A. fulgidus, H. marismortui and P. aerophilumhave all been cloned, functionally expressed, and characterized in E.coli (Musfeldt et al., supra; Brasen et al., supra (2004)).

Gene Accession No. GI No. Organism AF1211 NP_070039.1 11498810Archaeoglobus fulgidus DSM 4304 scs YP_135572.1 55377722 Haloarculamarismortui ATCC 43049 PAE3250 NP_560604.1 18313937 Pyrobaculumaerophilum str. IM2

EXAMPLE XXIII Production of BDO Utilizing Carboxylic Acid Reductase

This example describes the generation of a microbial organism thatproduces 1,4-butanediol using carboxylic acid reductase enzymes.

Escherichia coli is used as a target organism to engineer the pathwayfor 1,4-butanediol synthesis described in FIG. 58. E. coli provides agood host for generating a non-naturally occurring microorganism capableof producing 1,4-butanediol. E. coli is amenable to genetic manipulationand is known to be capable of producing various products, like ethanol,acetic acid, formic acid, lactic acid, and succinic acid, effectivelyunder various oxygenation conditions.

Integration of 4-Hydroxybutyrate Pathway Genes into Chromosome:Construction of ECKh-432. The carboxylic acid reductase enzymes wereexpressed in a strain of E. coli designated ECKh-761 which is adescendent of ECKh-432 with additional deletions of the sad and gabDgenes encoding succinate semialdehyde dehydrogenase enzymes. This straincontained the components of the BDO pathway, leading to 4HB, integratedinto the chromosome of E. coli at the fimD locus as described in ExampleXXI.

Cloning and Expression of Carboxylic Acid Reductase and PPTase. Togenerate an E. coli strain engineered to produce 1,4-butanediol, nucleicacids encoding a carboxylic acid reductase and phosphopantetheinetransferase are expressed in E. coli using well known molecular biologytechniques (see, for example, Sambrook, supra, 2001; Ausubel supra,1999). In particular, car genes from Nocardia iowensis (designated 720),Mycobacterium smegmatis mc(2)155 (designated 890), Mycobacterium aviumsubspecies paratuberculosis K-10 (designated 891) and Mycobacteriummarinum M (designated 892) were cloned into pZS*13 vectors (Expressys,Ruelzheim, Germany) under control of PA1/lacO promoters. The npt(ABI83656.1) gene (i.e., 721) was cloned into the pKJL33S vector, aderivative of the original mini-F plasmid vector PML31 under control ofpromoters and ribosomal binding sites similar to those used in pZS*13.

The car gene (GNM_720) was cloned by PCR from Nocardia genomic DNA. Itsnucleic acid and protein sequences are shown in FIGS. 59A and 59B,respectively. A codon-optimized version of the npt gene (GNM_721) wassynthesized by GeneArt (Regensburg, Germany). Its nucleic acid andprotein sequences are shown in FIGS. 60A and 60B, respectively. Thenucleic acid and protein sequences for the Mycobacterium smegmatismc(2)155 (designated 890), Mycobacterium avium subspeciesparatuberculosis K-10 (designated 891) and Mycobacterium marinum M(designated 892) genes and enzymes can be found in FIGS. 64, 65, and 66,respectively. The plasmids were transformed into ECKh-761 to express theproteins and enzymes required for 1,4-butanediol production.

Demonstration of 1,4-BDO Production Using Carboxylic Acid Reductase.Functional expression of the 1,4-butanediol pathway was demonstratedusing E. coli whole-cell culture. Single colonies of E. coli ECKh-761transformed with the pZS*13 and pKJL33S plasmids containing a car geneand GNM_721, respectively, were inoculated into 5 mL of LB mediumcontaining appropriate antibiotics. Similarly, single colonies of E.coli ECKh-761 transformed with car-containing pZS*13 plasmids andpKJL33S plasmids with no insert were inoculated into additional 5 mLaliquots of LB medium containing appropriate antibiotics. Ten mLmicro-aerobic cultures were started by inoculating fresh minimal in vivoconversion medium (see below) containing the appropriate antibioticswith 1.5% of the first cultures.

Recipe of the minimal in vivo conversion medium (for 1000 mL) is asfollows:

Final concentration 1M MOPS/KOH buffer 100 mM Glucose (40%) 1% 10XM9salts solution 1X MgSO4 (1M)  1 mM trace minerals (×1000) 1X 1M NaHCO3 10 mM

Microaerobic conditions were established by initially flushing cappedanaerobic bottles with nitrogen for 5 minutes, then piercing the septumwith an 18G needle following inoculation. The needle was kept in thebottle during growth to allow a small amount of air to enter thebottles. Protein expression was induced with 0.2 mM IPTG when theculture reached mid-log growth phase. This is considered: time=0 hr. Theculture supernatants were analyzed for BDO, 4HB, and other by-productsas described above and in WO2008115840 (see Table 31).

Table 33 shows the production of various products in the strainsexpressing various carboxylic acid reductases, including production ofBDO.

TABLE 33 Production of various products in strains expressing variouscarboxylic acid reductases. Cm10 Carb100 Carb100 0 h Strain pKLJ33SpZS*13S pZShc13S OD600 OD600 1 761 034rbs55 no insert 0.54 2.13 5 761721 720 0.48 1.88 7 761 721 890 0.45 1.63 8 761 721 891 0.48 1.65 9 761721 892 0.45 1.31 12 761 no insert 720 0.50 1.72 14 761 no insert 8900.51 1.96 15 761 no insert 891 0.19 2.36 16 761 no insert 892 0.05 1.4048 h PA Su La 4HB BDO GBL EtOH_(Enz) 48 h, mM 1 10.60 0.00 0.20 8.082.40 2.97 0.65 5 3.41 0.00 0.02 6.93 8.53 0.24 1.82 7 0.00 0.00 0.006.26 12.30 0.47 5.85 8 2.16 0.00 0.00 7.61 9.08 0.46 2.84 9 0.36 0.000.00 5.89 7.83 0.15 2.89 12 8.30 0.00 0.13 9.91 1.99 0.14 0.64 14 2.570.00 0.01 9.77 3.53 0.14 1.44 15 1.73 0.00 0.00 9.71 2.68 0.10 0.79 160.02 0.00 0.00 10.80 1.30 0.07 0.55 48 h, mM/OD 1 4.98 0.00 0.09 3.801.13 1.40 0.31 5 1.81 0.00 0.01 3.69 4.54 0.13 0.97 7 0.00 0.00 0.003.84 7.55 0.29 3.59 8 1.31 0.00 0.00 4.61 5.50 0.28 1.72 9 0.27 0.000.00 4.50 5.99 0.12 2.21 12 4.83 0.00 0.07 5.76 1.16 0.08 0.37 14 1.310.00 0.01 4.99 1.80 0.07 0.74 15 0.73 0.00 0.00 4.11 1.13 0.04 0.33 160.01 0.00 0.00 7.71 0.93 0.05 0.39 PA = pyruvate, SA = succinate, LA =lactate, 4HB = 4-hydroxybutyrate, BDO = 1,4-butanediol, GBL =gamma-butyrolactone, Etoh = ethanol, LLOQ = lower limit ofquantificationThese results show that various carboxylic acid reductases can functionin a BDO pathway to produce BDO.

EXAMPLE XXIV Metabolic Modifications to Improve Engineered MicroorganismCharacteristics

This example describes additional metabolic modifications to improvecharacteristics of an engineered microorganism. Such improvedcharacteristics include, but are not limited to, increased productyield, decreased production of by-products, improved growthcharacteristics of the engineered microorganisms, including improvedcharacteristics for scale up production, and the like.

Cultivation conditions for bottles. Twenty-milliliter bottlecultivations for metabolite production or bioconversion were performedin M9 minimal salts medium (6.78 g/L Na₂HPO₄, 3.0 g/L KH₂PO₄, 0.5 g/LNaCl, 1.0 g/L NH₄Cl, 1 mM MgSO₄, 0.1 mM CaCl₂) supplemented with 10 mMNaHCO₃, 20 g/L D-glucose and 100 mM MOPS to improve the bufferingcapacity, 10 μg/ml thiamine and the appropriate antibiotics for plasmidmaintenance. E. coli strain MG1655 lacIQ+ was grown anaerobically, andanaerobic conditions were obtained by flushing capped anaerobic bottleswith nitrogen for at least 5 min. Microaerobic conditions were used forall other strains, which were established by initially flushing cappedanaerobic bottles with nitrogen for 5 min, then piercing the septum witha 23G needle (Becton-Dickenson) after inoculation. The needle was keptin the bottle during growth to allow a small amount of air to enter.Protein expression was induced with 0.2 mM IPTG when the culture reachedmid-log growth phase, unless otherwise indicated in the text.

Cultivation conditions for 96 well-plates. All the cultures in 96well-plates were grown in 1.2 ml of M9 medium (composition providedabove) with MOPS and appropriate antibiotics. Carbon source in the formof 5% glucose was also added. Microaerobic conditions were obtained bycovering the plates with two gas-permeable adhesive seals. The edges ofthe seal were taped to minimize evaporation. All the cultures were grownat 37° C.

Bacterial strains and plasmids. Genomic DNA from bacterial strains wasisolated with the PureLink Genomic DNA Mini Kit (Invitrogen) accordingto the manufacturer's instructions. Recombinant DNA manipulations wereconducted as described (Sambrook et al., T. Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., (1989). Genes and open reading frames in the study were amplifiedfrom appropriate genomic DNA templates using the high-fidelity KOD DNApolymerase enzyme (EMD Chemicals; Billerica Mass.). Analytical PCRexperiments for genotyping and sequencing were conducted according tostandard molecular biology protocols with Taq polymerase (Promega). DNAsequencing was provided by Genewiz (South Plainfield N.J.).

Procedure for genetic manipulations. Development of expression vectorsfor BDO pathway. Vector backbones were obtained from Dr. Rolf Lutz ofExpressys (expressys.de). The vectors and strains are based on the pZExpression System described previously (Lutz and Bujard, Nucleic AcidsRes. 25:1203-1210 (1997)). Vectors obtained were pZE13luc, pZA33luc,pZS*13luc and pZE22luc and contained the luciferase gene as a stufferfragment. To replace the luciferase stuffer fragment with a lacZ-alphafragment flanked by appropriate restriction enzyme sites, the luciferasestuffer fragment was first removed from each vector by digestion withEcoRI and XbaI. The lacZ-alpha fragment was PCR amplified from pUC19with the following primers:

lacZalpha-RI (SEQ ID NO: 1)5′GACGAATTCGCTAGCAAGAGGAGAAGTCGACATGTCCAATTCACTGGCCGTCGTTTT AC3′lacZalpha 3′BB (SEQ ID NO: 2)5′-GACCCTAGGAAGCTTTCTAGAGTCGACCTATGCGGCATCAGAGCAGA-3′.

This generated a fragment with a 5′ end of EcoRI site, NheI site, aRibosomal Binding Site, a SalI site and the start codon. The 3′ end ofthe fragment contained the stop codon, XbaI, HindIII, and AvrII sites.The PCR product was digested with EcoRI and AvrII and ligated into thebase vectors digested with EcoRI and XbaI (XbaI and AvrII havecompatible ends and generate a non-site). Because NheI and XbaIrestriction enzyme sites generate compatible ends that can be ligatedtogether (but generate a site after ligation that is not digested byeither enzyme), the genes cloned into the vectors could be “Biobricked”together (openwetware.org/wiki/Synthetic_Biology:BioBricks). Briefly,this method allows joining an unlimited number of genes into the vectorusing the same 2 restriction sites, as long as the sites do not appearinternal to the genes, because the sites between the genes are destroyedafter each addition. Initially, expression was low from these vectors,and they were subsequently modified using the Phusion® Site-DirectedMutagenesis Kit (NEB, Ipswich, Mass.) to insert the spacer sequenceAATTAA between the EcoRI and NheI sites. This eliminated a putative stemloop structure in the RNA that bound the RBS and start codon.

All vectors have the pZ designation followed by letters and numbersindicating the origin of replication, antibiotic resistance marker andpromoter/regulatory unit. The origin of replication is the second letterand is denoted by E for ColE1, A for p15A and S for pSC101 (as well as alower copy number version of pSC101 designated S*)—based origins. Thefirst number represents the antibiotic resistance marker (1 forAmpicillin, 2 for Kanamycin, 3 for Chloramphenicol). The final numberdefines the promoter that regulated the gene of interest (1 forPLtetO-1, 2 for PLlacO-1 and 3 for PA1lacO-1) and each of thesepromoters became activated by its corresponding inducer molecule (pLtetOcan be induced by tetracycline; pLlacO-1 and pA1lacO-1 can be induced byIPTG). Three base vectors, pZS*13S, pZA33S and pZE13S, were thendesigned and constructed to serve as “inducible” plasmid vectors.

In addition to the “inducible” promoters mentioned above, a set of“constitutive” promoters were sampled from the Registry(partsregistry.org). Each of these “constitutive” promoters was thenintroduced into the pZS*13S vector backbone to replace the pA1lacO-1inducible promoter via Sequence and Ligation Independent Cloning (SLIC)method described by L1 & Eledge (Nature Methods 2007, 4:251-256). Ofthese sampled “constitutive” promoters (p100, p104, p105, p107, p108,p111, p115 & p119), experiments were carried out to establish an orderof promoter strength that was verified by protein expression levels. Forthese experiments, both “inducible” and “constitutive” plasmid vectorswere employed, modified for the biobricks and SLIC insertions asdiscussed above. To further fine-tune protein expression levels of someoverly expressed proteins, ribosomal binding site (RBS) in betweenpromoter and gene coding sequence was modified accordingly using the RBScalculator (salis.psu.edu/software/).

The SLIC primers used for inserting a list of genes into the pZS*13Svector backbone are listed below. The lower case marks the sequencesannealing to a vector backbone while the upper case marks the sequencesannealing to the coding region of a gene.

1. ppc 525 (SEQ ID NO: 100)Forward SLIC primer: gaggagaagtcgacATGAACGAACAATATTCCGCATTGCG(SEQ ID NO: 101)Reverse SLIC primer: ggaagctttctagaTTAGCCGGTATTACGCATACCTGCC2. sucAB-lpdA (SEQ ID NO: 102)Forward SLIC primer: taagctagcaagaggagaagtcgacATGCAGAACAGCGCTTTGAAAG(SEQ ID NO: 103)Reverse SLIC primer: gcctctaggaagctttctagaTTACTTTTTCTTCGCTTTGGCG3. galP 1120 (SEQ ID NO: 104)Forward SLIC primer: ctagcaagaggagaagtcgacATGCCTGACGCTAAAAAACAGGGGCGGT(SEQ ID NO: 105)Reverse SLIC primer: ctaggaagctttctagagtcgTTAATCGTGAGCGCCTATTTCGCGCAG4. glk 1123 (SEQ ID NO: 106)Forward SLIC primer: ctagcaagaggagaagtcgacATGACAAAGTATGCATTAGTCGGTGATGTG(SEQ ID NO: 107)Reverse SLIC primer: ctaggaagctttctagagtcgTTACAGAATGTGACCTAAGGTCTGGCGTAAATG5. glf 1786 (SEQ ID NO: 108)Forward SLIC primer: ctagcaagaggagaagtcgacATGAGTTCTGAAAGTAGTCAGGGTCTAGTC(SEQ ID NO: 109)Reverse SLIC primers: ctaggaagctttctagagtcgTTACTTCTGGGAGCGCCACATCTC

For the work discussed below, three base vectors, pZS*13S, pZA33S andpZE13S, modified for the biobricks insertions as discussed above, wereemployed.

The following technique describes the construction of the incW plasmids,pPSX13 and pPSX23R. The original incW plasmid backbone (pPSX) wasobtained from Dr. John M. Pemberton (Sarovich and Pemberton, Plasmid57:306-313 (2007)). The pPSX plasmid was modified to insert either anampicillin marker (bla gene), designated as pPSX13, into a BamHIrestriction enzyme site or an kanamycin marker and a R6k replicationorigin, designated as pPSX23R, in between BamHI and Sad restrictionenzyme sites. Two similar incW plasmids were also constructed to includethe ald-adh gene cassette amplified from pZS*13S-ald-adh. ForpPSX13-ald-adh ampicillin-resistant plasmid, the bla-ald-adh genecassette was inserted into a BamHI restriction enzyme site of pPSX13. Toconstruct pPSX23R-ald-adh kanamycin resistant plasmid, the ampicillinmarker (bla gene) was replaced with the “kanamycine marker-R6kreplication origine” cassette in between XhoI and Sad restriction enzymesites of pPSX23R.

The genes 033B, 1210, and 956 were inserted in the mini-F-plasmidbackbone pKLJ33S as follows: PCR primers were designed to amplify thegenes 033B (033BFOR and 033BREV), 1210C (1210CFOR and 1210CREV), and 956(956FOR and 956REV) from appropriate templates. These primers includedthe immediate 5′ (FOR) and 3′ (REV) ends of each gene as denoted inupper case. Each primer also included a tail that was directlyhomologous to the promoter region (FOR) and terminator region (REV) ofplasmid pZA33S. A linear fragment of pZA33S with the promoter region andterminator regions at either extreme end was amplified using PrimerspZ-5′ and pZ-3′ and circular pZA33S as a template. The pZA33S linearfragment was combined with each of the 033B, 1210C, and 956 genefragments and joined using sequence- and ligation-independent cloning toform the corresponding plasmids pZA33S-033B, pZA33S-1210C, andpZA33S-956.

Primer SEQ ID Designation Primer Sequence NO 033BFORtagcaagaggagaagtcgacATGGACTGGAAGAAGATCTATGAAG 110 033BREVcctctaggaagctttctagaTTAGAATGCCGCGTTGAAG 111 1210CFORtagcaagaggagaagtcgacATGAGCTGGCAAGAACTGTATC 112 1210CREVcctctaggaagctttctagaTTAATATTTCTCTTTAAAGCGCTTTTC 113 956FORtagcaagaggagaagtcgacATGGCACGTTTTACTTTACCAAG 114 956REVcctctaggaagctttctagaTTACAAATTAACTTTAGTTCCATAGTATGTGC 115 034Cmforaccggtaaacgactcagcagcctgacgcactggcgatattattttgccttctcctcaccacagaatgttct116 gccacctgaCGATATCAAATTACGCCCCG 034Cmrevtgcttatccacaacattttgcgcacggttatgtggacaaaatacctggttacccaggccgtgccggcacgt117 taaccgggcCCTAGGTCTAGGGCGGCGGATTTG

Primers 034 Cmfor and 034 Cmrev were used to amplify the 033B, 1210C,and 956 genes from the appropriate pZA33S plasmid background. Theseprimers were designed to amplify the chloramphenicol resistance gene,pA1 promoter, particular gene of interest, and terminator elements fromplasmid pZA33S (with the sequences denoted in upper case) while tailsequences (denoted in lower case) corresponded to regions flanking butnot including the ampicillin resistance gene, arabinose responsivepromoter and terminator sequences of the mini-F-plasmid pKLJ12 (Jonesand Keasling, Biotechnol. Bioengineer. 59:659-665 (1998)).

An E. coli DP10B strain containing plasmid pKLJ12 andtemperature-sensitive Red/ET helper plasmid pREDET (Tet) (GeneBridgesGmbH, Heidelberg Germany) was inoculated into LB medium containingtetracycline at 10 μg/ml and ampicillin at 100 μg/ml finalconcentrations and incubated for 3 hours at 30° C. with shaking untilcells were turbid. Arabinose to induce expression of the Red/ETrecombination functions was added to a final concentration of 0.3%, thetemperature was shifted to 37° C. and the cells were allowed to incubatefor an additional hour with shaking After being made electrocompetent byrepeated centrifugation and resuspension in ice-cold steriledouble-distilled water, these cells were mixed with 034 Cmfor/034 CmrevPCR amplifications from the pZA33S-033B, pZA33S-1210C, or pZA33S-956plasmids in a 1 mm cuvette and then subjected to electroporation at 1800kV, 25 μF, 200Ω. Electroporated cells were then added to SOC growthmedium, incubated at 37° C. without shaking, and then spread on LB solidmedium containing chloramphenicol at 10 μg/ml final concentration andincubated at 37° C. for 18 to 24 hours. Resultingchloramphenicol-resistant single colonies were then scored for growth onsolid LB media containing either tetracycline at 10 μg/ml or theampicillin at 100 μg/ml. Colonies that were shown to be chloramphenicolresistant, tetracycline sensitive, and ampicillin sensitive wereverified by PCR and DNA sequencing to contain new mini-F-plasmids thathad completely exchanged the ampicillin resistance gene/arabinoseresponsive promoter/terminator of pKLJ12 with the chloramphenicolresistance gene/PA1 promoter/gene-of-interest/terminator of pZA33S byRED/ET-mediated homologous recombination, resulting in the newmini-F-plasmids pKLJ33S-033B, pKLJ33S-1210C, pKLJ33S-956.

Chromosomal replacement of genes using the sacB gene. The primary methodused for the insertion or deletion of genes in the chromosome of E. coliwas based on the utilization of the sacB gene from Bacillus subtilis(Gay et al., J. Bacteriol. 153:1424-1431 (1983)). The vector used ispRE118 (ATCC87693) deleted of the oriT and IS sequences. The resultingvector (3.6 kb in size and carrying the kanamycin resistance gene) wassequenced and called pRE118-V2. All cloning of fragments was done intothe restriction sites KpnI and PstI of pRE118-V2, unless notified. AllPCR amplification used genomic DNA from E. coli MG1655 (ATCC47076) asDNA template. The first integration event in the chromosome was selectedon LB agar plates containing Kanamycin (25 or 50 mg/L). Correctinsertions were verified by PCR using 2 primers: one located outside theregion of insertion and one in the kanamycin gene(5′-aggcagttccataggatggc-3′; SEQ ID NO:118). Clones with the correctinsertion were selected for resolution. They were sub-cultured twice inplain liquid LB at the desired temperature and serial dilutions wereplated on LB-no salt-sucrose 10% plates. Clones that grew on sucrosecontaining plates were screened for the loss of the kanamycin resistancegene on LB-low salt agar medium and the deletion/insertion of thefragment of interest was verified by PCR and sequencing of theencompassing region.

This method was used for deleting appCB using the following primers:

SEQ Primer ID Designation Primer Sequence NO appCB Kpn fwAtaataataggtaccggcggcgctggcgcagcttgctgcg 119 appCB BamHI rvTATTATTATGGATCCAACCCGATAATGGTAGATCTCCCTCT 120 appCB BamHI fwAtaataataggatccggagcagaaacaatgtggtatttact 121 appCB Pst rvTATTATTATCTGCAGATGCTCTTTTTTATGCATTACAAACTGC 122

As a result, the following nucleotides were deleted:1,036,963->1,039,655

tesB and ybgC deletions were also made with aforementioned pRE-sacB-Kanvector. The primers used for making the deletion were: (5′-3′)

Primer Designation Primer Sequence SEQ ID NO tesB-kpn-frwtaataataaggtaccaactgggcttgcttcactgg 123 tesB-bgl-revtaataataaAGATCTGCCTCGACCGTTCAGGAAGG 124 tesB-bgl-frwtaataataaagatcttaactctccagtaacaaagctgc 125 tesB-Pst-revtaataataaCTGCAGGCTATGTCACCACTTACGG 126 ybgC-Pst-frwTattattattcctgcaggcgtctttgttcttccgtcc 127 ybgC-Kpn-revtattattatCTTCGGTACCTTTAGCATCTGCTTCGGCC 128 ybgC-Bam-revtattattatGGATCCCGGTAATGCAACAAAAGTTAGAGC 129 ybgC-Bam-frwtattattatggatccaaagtgactgacatgaatatccttgatttgttc 130

Nucleotides deleted as a consequence were:

-   tesB: 473,525<-474,385-   ybgC: 773,975->774,379

An ndh deletion was also made with the standard pRE-sacB-Kan vector. Theprimers used for making the deletion were: (5′-3′)

Primer Designation Primer Sequence SEQ ID NO ndh-Bam-frwtaataataaggatcctacactggcggatgtggcataaac 131 ndh-Kpn-revtaataatttggtacccATTCACAGTCACCAGGTACAACG 132 ndh-Bam-revtaataataaGGATCCGAGAATAACATGAATGGTGCATTG 133 ndh-Pst-frwtaataatatctgcagatccacaaaaagccctggcaattg 134

Nucleotides deleted as a result of the deletion were:1,165,308->1,166,612

Deletions of yjgB, yqhD, yahK and adhP were introduced with the standardpRE-sacB-Kan vector. The primers used for making the deletion were:(5′-3′)

Primer Designation Primer Sequence SEQ ID NO ygjB-Pst-frwgataatcccccctgcagggagcggtaaatag 135 ygjB-Bam-revtattatTTTGGATCCTTCTCTGGTGTTGTTTGGG 136 yjgB-Bam-frwtattattatggatccgcgtggtgttgaaagccg 137 yjgBKpn-revtattatAAAGGTACCGCCTCTTCCAGGTCAGTGAAGGG 138 yqhD-Kpn-frwtaataataaggtacccagttttggctatgccttaag 139 yqhD-Hind-revtaataaTCATTAAGCTTGCTCCCTTTGCTGGGCC 140 yqhD-Hind-frwtaataataataagctttttacgcctcaaactttcg 141 yqhD-Pst-revtaataataaATTGCCCTGCAGCGTAAGATTGTCGTTCAGGG 142 yahK-Kpn-frwtattattattgtttggtacctctgtgccgct 143 yahK-Pst-revAATGTTCCACTCTGCAGGGATGATAATAAGGGG 144 yahK-Bam-frwatcggatccaatcgcacactaacagactg 145 yahK-Bam-revACAGCTTTGGATCCTCATTGTGTTTACTCCTGATTAGC 146 adhP XbaI fwataataatatctagagcagcaagccgcgcggcaggtggtcag 147 adhP PstI rvTATTATTATCTGCAGACGCCATCCTGATCCATATGTATATGG 148 adhP Bam upTATTATTATGGATCCAGTTCCTCCTTTTCGGATGATGTTCTG 149 rv adhP Bamataataataggatccgaggcctttgctgcgactgccatgttc 150 down fw

Nucleotides deleted as a result of each of these deletions were: ygjB:4,493,213<-4,494,232, yqhD: 3,153,377->3,154,540, yahK:342,108->343,157, adhP: 1,550,852<-1,551,862.

Gene deletions using lambda-red mediated combination: Native genescyoABCD were deleted from the chromosome by Red-mediated recombination(ncbi.nlm.nih.gov/pubmed/10829079).

For cyoABCD deletion, the following primers were used to amplify achloramphenicol or kanamycin resistance gene from pKD3 or pKD4(ncbi.nlm.nih.gov/pubmed/10829079), respectively, and were flanked byFRT sites and homologies to cyoABCD:

LK-cyo-F1 (SEQ ID NO: 151) CGCCACAACCAGTGACACCC LK-cyo-R1(SEQ ID NO: 152) GGGGTTTTAGTCGCCCTTTCTGGC LK-cyo-IO-down(SEQ ID NO: 153) CGGGATCTGGTGGCGTTTAAAGTG LK-cyo-IO-up (SEQ ID NO: 154)TGACGGCGTTTGTCTTCACCG

E. coli K-12 MG1655 carrying the Red helper plasmid pKD46 was grown in100 ml LB medium with ampicillin and 1 mM L-arabinose at 30° C. to anOD600 of 0.3, and electroporation-competent cells were prepared asdescribed elsewhere (Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,(1989)). A measure of 50 μl of competent cells was mixed with 400 ng ofthe PCR fragment in an ice-cold 0.1 cm cuvette (Bio-Rad Inc., Hercules,Calif., USA). Cells were electroporated at 1.8 kV with 25 mF and 200,immediately followed by the addition of 1 ml of SOC medium (2% BactoTryptone (Difco), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10mM MgCl₂, 10 mM MgSO₄, 20 mM glucose) with 1 mM L-arabinose. Afterincubation for 2 h at 37° C., one-tenth portion was spread onto agarplate to select chloramphenicolR or kanamycinR transformants at 37° C.The individual deletions were then transduced into new host strainsusing P1vir and selecting for the appropriate antibiotic (Silhavy etal., Experiments with gene fusions, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1984)). The chloramphenicol or kanamycin resistantcolonies were then transformed with the temperature sensitive plasmidpCP20, and plated on ampicillin (100 μg/ml) plates at 30° C. pCP20contains the yeast Flp recombinase gene, chloramphenicol and ampicillinresistant genes, and temperature sensitive replication. Colonies weregrown in LB medium without antibiotics at 42° C. overnight and streakplated on prewarmed LB Agar plates and grown at 42° C. Individual cloneswere tested for sensitivity to ampicillin and chloramphenicol orkanamycin to isolate clones which had lost the pCP20 plasmid and had thechloramphenicol or kanamycin resistance gene lost via the Flprecombinase. Positive clones with deletions in cyoABCD contain a singleFRT “scar” and were verified by PCR using primers which flanked theirrespective genes.

The nucleotides deleted because of the cyoABCD deletion were:449,887->447,270

The esterase from Yersinia intermedia was integrated in the chromosomewith the standard pRed technique described above. The primers used formaking the insertion were: (5′-3′)

Primer Designation Primer Sequence SEQ ID NO JE310ACGCTATGGAACTGCAGGATGAGAGCATGAAATGGAACGG 155 ATGCGCGCCTTAT JE311ACGCTATGGAACTGCAGGATGAGAGCATCTGCCCCAGATC 156 GTTGGCTTTTTGC JE312TTTGAAAACAGGATGTAGCGATGACCGATATTCGCTTGTA 157 JE312-rvTACAAGCGAATATCGGTCATCGCTACATCCTGTTTTCAAA 158 JE313CGCCTGAATACTACGATTGACTGGAGAATGCGAATGAACA 159 JE313-rvTGTTCATTCGCATTCTCCAGTCAATCGTAGTATTCAGGCG 160 JE314TTTGAAAACAGGATGTAGCGGCCCGTAGTCTGCAAATCC 161 JE314-rvGGATTTGCAGACTACGGGCCGCTACATCCTGTTTTCAAA 162 JE315GCATTACGCTGACTTGACGGCTGGAGAATGCGAATGAACA 163 JE315-rvTGTTCATTCGCATTCTCCAGCCGTCAAGTCAGCGTAATGC 164

The sequence that was inserted at the gabD locus (ORF swap) was:

(SEQ ID NO: 165) ACTGGAGAATGCGAATGAACAGCAATAAAGAGTTAATGCAGCGCCGCAGTCAGGCGATTCCCCGTGGCGTTGGGCAAATTCACCCGATTTTCGCTGACCGCGCGGAAAACTGCCGGGTGTGGGACGTTGAAGGCCGTGAGTATCTTGATTTCGCGGGCGGGATTGCGGTGCTCAATACCGGGCACCTGCATCCGAA GGTG.

Markerless deletion of sucC and sucD. Deletion of the native Escherichiacoli sucC (gene 2) and sucD (gene 3) from the chromosome (FIG. 67) wasachieved using a two-step double-crossover homologous recombinationmethod. Recombination was catalyzed via expression of the Lambda phageRed genes from pRED-Amp (Gene Bridges, Heidelberg, Germany). Genesencoding levansucrase (gene 6) and kanamycin resistance (gene 7) wereintegrated into the chromosome, replacing the majority of sucC (gene 2)and sucD (gene 3). Kanamycin was used to select for successfulintegrants. In a second double-crossover homologous recombination step,the integrated sequence containing the levansucrase gene (gene 6) andkanamycin resistance gene (gene 7) was replaced with an appropriate DNAsequence lacking the majority of sucC (gene 2) and sucD (gene 3), andsucrose resistant clones were selected for.

Linear double-stranded DNA sequences used in the two homologousrecombination steps outlined above were constructed via standardmolecular biological techniques. To make the sequence used in theinitial integration step, a 1100 bp sequence from within sucB (gene 1)through the 69th base pair of sucC (gene 2) was amplified by PCR fromEscherichia coli MG1655 genomic DNA using oligonucleotide 1 andoligonucleotide 3 (FIG. 68a ). The genes encoding levansucrase (gene 6)and kanamycin resistance (gene 7) were amplified by PCR from plasmidpRE-118 DNA using oligonucleotide 5 and oligonucleotide 6 (FIG. 68b ).Another 1100 bp sequence from the 811th by of sucD (gene 3), spanningmngR (gene 4) and part of mngA (gene 5) was amplified by PCR fromEscherichia coli MG1655 genomic DNA using oligonucleotide 4 andoligonucleotide 2 (FIG. 68a ). 20 by of oligonucleotide 3 overlaps witholigonucleotide 5 and 20 bp of oligonucleotide 6 overlaps witholigonucleotide 4, such that the three overlapping PCR products can becombined and amplified using standard procedures (FIG. 68c ). Likewise,the linear double-stranded DNA sequences used in the secondrecombination for deletion of sucCD was made by combining and amplifyingtwo overlapping PCR products. A 1100 bp sequence from within sucB(gene 1) through the 69th base pair of sucC (gene 2) was amplified byPCR from Escherichia coli MG1655 genomic DNA using oligonucleotide 1 andoligonucleotide 8 (FIG. 68d ). Another 1100 bp sequence from the 811thby of sucD (gene 3), spanning mngR (gene 4) and part of mngA (gene 5)was amplified by PCR from Escherichia coli MG1655 genomic DNA usingoligonucleotide 7 and oligonucleotide 2 (FIG. 68d ). Oligonucleotide 7overlaps with oligonucleotide 8, such that the two overlapping PCRproducts can be combined and amplified using standard procedures (FIG.68e ). The nucleotides deleted because of the sucCD deletion are from:762,306->764,320.

Oligo- nucleotide SEQ ID NO 1 GGCGAAAGAGTCTGCTCCGGC 166 2CATTCAGACACAACGCATCGCGG 167 3 GGCTTACCAGCACCGGTGGGTTATGCCCGTAGTCTGCAAATC168 C 4 GCATTACGCTGACTTGACGGGTGAAAACCGTTCGCAGCCTGG 169 5GGCTTACCAGCACCGGTGGGTTATGCCCGTAGTCTGCAAATC 170 C 6CCAGGCTGCGAACGGTTTTCACCCGTCAAGTCAGCGTAATGC 171 7GGCTTACCAGCACCGGTGGGTTATGTGAAAACCGTTCGCAGC 172 CTGG 8CCAGGCTGCGAACGGTTTTCACATAACCCACCGGTGCTGGTA 173 AGCC

Analytical Procedures: HPLC, LCMS/MS and GCMS were employed for analysisof cell culture and fermentation samples. Good correlation between thetwo techniques (variation within 10%) provided method cross-validationand ensured data accuracy.

Organic acids, acetate, pyruvate, lactate and succinate, as well as BDO,were measured by HPLC (Agilent 1100) equipped with diode array andrefractive index (R1) detectors, using an ion exclusion Carbomix H-NP5column (Sepax) with 5 mM sulfuric acid as the mobile phase at 0.6 mL/minflow rate, and 55° C. column temperature. Organic acids were monitoredin both UV absorption at 210 nm and refractive index detector, whileBDO, ethanol and simple sugars were measured using R1 only. Allchemicals and reagents were from Sigma-Aldrich (St. Louis, Mo., USA).

LCMS/MS analysis was performed on API3200 triple quadrupole system (ABSciex, Life Technologies, Carlsbad, Calif.), interfaced with Agilent1200 HPLC, utilizing electrospray ionization and MRM based acquisitionmethods. BDO, 4HB, GBL, Glu, Ala and GABA and internal standards (¹³Cisotopically labeled analogs were used for each analyte, respectively,to compensate for the matrix effects and ensure linearity) weremonitored in positive ionization mode. Negative ionization was used,when desired, for quantitation of acidic analytes, for example,pyruvate, lactate, succinate and 4HB. Chromatographic separation wasconducted on Zorbax Eclipse XDB C18 4.6×30 mm, 1.8 μm particle size,maintaining column at 45° C., flow rate 0.7 mL/min. Injection volume was5 uL. Eluents consisted of water with 0.1% formic acid and methanol with0.1% formic acid, analytes of interest eluted under isocratic conditionsof 5% methanol followed by the step gradient to 95% methanol, resultingin 2 min long LCMS method. Filtered samples were diluted in water,containing internal standards, dilution factor varied from 200 to 10,000depending on concentrations of metabolites of interest. Dilution inwater of 100-fold of greater resulted in no ion suppression effectsobserved for media composition and conditions used in 1,4-Butanediolproduction in E. coli (Yim et al., Nature Chemical Biology 7, 445-452,(2011)). Quantitation dynamic ranges were from 0.05-200 uM for BDO andother analytes. Quantitation was performed using Analyst software.LCMS/MS approach was a preferred method for monitoring BDO, 4HB, GBL,Glu, Ala and GABA due to its fast turnaround time allowing highthroughput operation.

In GCMS approach, BDO, 4HB, lactate and succinate in culture brothsamples were derivatized by trimethylsilylation and quantitativelyanalyzed by GCMS, using a procedure adapted from the literature withsome modifications. The developed method demonstrated sensitivity downto low μM level, linearity in 0.05-15 mM range, as well as goodreproducibility. 100 μL filtered samples were dried down in a speedvacconcentrator for approximately 1 hour at ambient temperature, followedby the addition of 20 μL 10 mM cyclohexanol solution, as an internalstandard, in dimethylformamide. 100 μLN,O-bis(trimethylsilyl)trifluoro-acetimide (BSTFA) with 1%trimethylchlorosilane was added, and the mixture was incubated at 70° C.for 30 min. The derivatized samples were centrifuged for 5 min anddirectly injected into GCMS (Agilent 6890N/5973N). An Rtx-5SIL MScapillary column (Restek, 30m×0.25 mm ID×0.25 μm film thickness) wasused. The GC was operated in a split injection mode introducing 1 μL ofsample at 20:1 split ratio. The injection port temperature was 250° C.Helium was used as a carrier gas, and the flow rate was maintained at1.0 mL/min. A temperature gradient program was optimized to ensure goodresolution of the analytes of interest and minimum matrix interference.The oven was initially held at 80° C. for 1.5 min, then ramped to 140°C. at 10° C./min and 3 min hold, followed by fast ramping to 300° C. at100° C./min and final hold for 5 min. The MS interface transfer line wasmaintained at 280° C. The total analysis time was 17 min including 4 minsolvent delay. Standard calibration curves were constructed usinganalyte solutions in the corresponding cell culture or fermentationmedium to match sample matrix as close as possible. Ethanol was assayedby GCMS as well, following sample dilution in methanol, using anHP-InnoWax capillary column (30m, 0.25 mm ID, 0.25 um film thickness).GCMS data were processed using ChemStation software (Agilent).

Residual glucose in the fermentation samples was measured using either aYSI instrument or HPLC-UV-RL Ethanol was measured by either YSI or GCMS.

The metabolic engineering strategies described herein were applied toincrease 1,4-butanediol production in Escherichia coli or otherwiseimprove characteristics of the engineered microorganisms. The employed1,4-butanediol pathway for these studies is depicted in FIG. 69. Thegenes tested for each of the six steps shown in FIG. 69 are described inTables 34, 38, 40, 42, 46 and 49.

EXAMPLE XXV Production of 4-Hydroxybutyrate and 1,4-Butanediol UponExpression of Genes Encoding Alpha-Ketoglutarate Decarboxylase Enzymes

This example describes production of 4-hydroxybutyrate (4HB or 4hb) and1,4-butanediol (BDO) in cells engineered to express genes encodingalpha-ketoglutarate decarboxylase enzymes.

Several heterologous alpha-ketoglutarate decarboxylase genes (Table 34)were expressed in E. coli from the pZS*-13S plasmid to allow afunctional pathway from alpha-ketoglutarate to 4-hydroxybutyrate and1,4-butanediol. Host strains include 3125, 2869, and 3812. These hoststrains are based on strain ECKh-432 whose construction is describedabove and in US20110045575. Notable modifications include deletions inadhE, ldhA, pflB, mdh, and arcA. Strain ECKh-432 also containschromosomally integrated copies of sucD from Porphyromonas gingivalis(encoding CoA-dependant succinate semialdehyde dehydrogenase), sucA fromMycobacterium bovis (encoding alpha-ketoglutarate decarboxylase), 4hbdfrom P. gingivalis (encoding 4-hydroxybutyrate dehydrogenase), and 4hbdfrom Clostridium kluyveri (encoding 4-hydroxybutyrate dehydrogenase).For this study, the M. bovis sucA and P. gingivalis sucD genes weredeleted in strains 3125, 2869, and 3812. Additionally, strains 3125,2869, and 3812 each contain a chromosomally integrated copy of4-hydroxybutyryl-CoA transferase expressed from the pA promoter at theattB locus. Strains 2869 and 3812 were tested with 4-hydroxybutyryl-CoAreductase and 4-hydroxybutyraldehyde reductase expressed from thepPSX23R plasmid. Tables 35, 36, and 37 show that expression ofheterologous alpha-ketoglutarate decarboxylase genes enhances 4hb and/orBDO production in strains 3125, 2869, and 3812, respectively, over thecontrols denoted by “pZS*13S”. Cells were cultured using the 96well-plate protocol described previously.

TABLE 34 Genes encoding functional alpha-ketoglutarate decarboxylaseenzymes. Gene Number Organism Accession GI 42 Mycobacterium tuberculosisZP_06504371.1 289744993 1717 Mycobacterium smegmatis YP_889299.1118472055 1720 Saccharopolyspora erythraea YP_001108480.1 134102819 1727Jonesia denitrificans YP_003161752.1 256833025 1715 Mycobacterium aviumsubsp. NP_961470.1 41408634 Paratuberculosis 1718 Nocardia farcinicaYP_120910.2 161598437 1726 Streptomyces avermitilis NP_824148.1 298295141727 Jonesia denitrificans YP_003161752.1 256833025 1913 Zymomonasmobilis YP_162422.1 56551583 1914 Pseudomonas putida NP_743318.126987893 1915 Mycobacterium avium subsp. EGO39007.1 336460099Paratuberculosis 1916 Streptomyces griseus YP_001827718.1 182439999 1917Methanosarcina acetivorans NP_616881.1 20090806 1918 Clostridiumljungdahlii YP_003782019.1 300857035 1920 Synechocystis sp. PCC 6803NP_441304.1 16330576 1921 Synechococcus sp. PCC 7335 ZP_05039761.1254426044 1922 Clostridium acetobutylicum NP_350234.1 15896885 1923Clostridium carboxidivorans ZP_05391417.1 255524461 P7

TABLE 35 BDO and 4-hydroxybutyrate (4HB) production of E. coli hoststrain 3125 containing several alternative alpha-ketoglutaratedecarboxylase plasmid constructs. The plasmid construct denoted by only“pZS*13S” contains no alpha-ketoglutarate decarboxylase gene. Plasmidconstructs denoted by pZS*13S followed by a number express thealpha-ketoglutarate decarboxylase gene denoted by such number. BDO and4HB concentrations are in mM. LCMS - 19 h LCMS - 19 h Host strainPlasmid OD 19 h 4HB BDO 3125 pZS*13S 3.61 0.208 0.327 3125 pZS*13S 2.010.259 0.082 3125 pZS*13S 3.39 0.208 0.359 3125 pZS*13S 3.38 0.178 0.3523125 pZS*13S 3.84 0.277 0.478 3125 pZS*13S 4.01 0.253 0.479 3125 pZS*13S3.88 0.283 0.422 3125 pZS*13S 3.54 0.192 0.499 3125 pZS*13S-1717 4.3017.300 1.520 3125 pZS*13S-1717 3.52 17.400 1.280 3125 pZS*13S-1717 3.7917.700 1.540 3125 pZS*13S-1717 3.53 17.800 1.550 3125 pZS*13S-1720 2.923.940 0.196 3125 pZS*13S-1720 1.58 2.140 0.037 3125 pZS*13S-1720 1.972.600 0.072 3125 pZS*13S-1720 3.11 3.870 0.261 3125 pZS*13S-1727 12.052.330 0.694 3125 pZS*13S-1727 3.23 3.040 1.320 3125 pZS*13S-1727 3.193.490 1.560 3125 pZS*13S-1727 4.86 2.500 0.657 3125 pZS*13S-1715 3.8914.900 1.880 3125 pZS*13S-1715 4.14 15.600 1.950 3125 pZS*13S-1715 4.0715.700 1.910 3125 pZS*13S-1715 3.03 14.300 1.600 3125 pZS*13S-1718 2.364.020 0.277 3125 pZS*13S-1718 1.82 4.630 0.406 3125 pZS*13S-1718 2.373.690 0.265 3125 pZS*13S-1718 2.26 3.940 0.273 3125 pZS*13S-1718 2.044.890 0.389 3125 pZS*13S-1718 2.42 4.720 0.338 3125 pZS*13S-1718 2.164.530 0.377 3125 pZS*13S-1718 2.43 5.190 0.517

TABLE 36 BDO and 4-hydroxybutyrate (4HB) production of E. coli hoststrain 2869 containing several alternative alpha-ketoglutaratedecarboxylase plasmid constructs. The plasmid construct denoted by only“pZS*13S” contains no alpha-ketoglutarate decarboxylase gene. Plasmidconstructs denoted by pZS*13S followed by a number express thealpha-ketoglutarate decarboxylase gene denoted by such number. BDO and4HB concentrations are in mM. LCMS - 16 h LCMS - 16 h Host strainPlasmid OD 16 h 4HB BDO 2869 pZS*13S 3.39 0.04 0.42 2869 pZS*13S 3.410.04 0.42 2869 pZS*13S 3.43 0.04 0.35 2869 pZS*13S 3.51 0.04 0.39 2869pZS*13S 3.51 0.03 0.39 2869 pZS*13S 3.50 0.04 0.40 2869 pZS*13S 3.550.03 0.36 2869 pZS*13S 3.50 0.02 0.35 2869 pZS*13S-042 3.50 0.33 2.812869 pZS*13S-042 3.52 0.32 2.69 2869 pZS*13S-042 3.58 0.32 3.05 2869pZS*13S-042 3.49 0.28 2.61 2869 pZS*13S-042 3.39 0.30 2.87 2869pZS*13S-042 3.41 0.28 3.06 2869 pZS*13S-042 3.44 0.27 2.64 2869pZS*13S-042 3.28 0.33 2.90 2869 pZS*13S-1726 2.74 0.34 2.27 2869pZS*13S-1726 2.78 0.23 2.75 2869 pZS*13S-1726 2.96 0.43 2.80 2869pZS*13S-1726 2.84 0.34 2.33 2869 pZS*13S-1727 3.43 0.06 2.39 2869pZS*13S-1727 2.79 0.07 2.18 2869 pZS*13S-1727 3.09 0.07 2.12 2869pZS*13S-1727 3.03 0.07 2.38

TABLE 37 BDO and 4-hydroxybutyrate (4HB) production of E. coli hoststrain 3812 containing several alternative alpha-ketoglutaratedecarboxylase plasmid constructs. The plasmid construct denoted by only“pZS*13S” contains no alpha-ketoglutarate decarboxylase gene. Plasmidconstructs denoted by pZS*13S followed by a number express thealpha-ketoglutarate decarboxylase gene denoted by such number. BDO and4HB concentrations are in mM. LCMS - 24 h LCMS - 24 h Host strainPlasmid OD 24 h 4HB BDO 3812 pZS*13S 3.75 0.07 1.71 3812 pZS*13S 3.740.05 1.46 3812 pZS*13S 3.49 0.04 2.08 3812 pZS*13S 3.59 0.05 2.31 3812pZS*13S-1913 3.40 6.51 9.25 3812 pZS*13S-1913 3.28 6.46 8.69 3812pZS*13S-1913 3.41 6.22 8.53 3812 pZS*13S-1913 3.25 5.94 7.89 3812pZS*13S-1914 3.83 23.10 85.00 3812 pZS*13S-1914 3.41 29.70 93.10 3812pZS*13S-1914 3.68 19.90 63.10 3812 pZS*13S-1914 3.45 27.20 101.00 3812pZS*13S-1915 4.17 0.30 3.28 3812 pZS*13S-1915 3.78 0.18 3.11 3812pZS*13S-1915 3.95 0.14 3.08 3812 pZS*13S-1915 3.97 0.11 2.80 3812pZS*13S-1916 4.09 0.07 4.82 3812 pZS*13S-1916 3.69 0.12 4.54 3812pZS*13S-1916 3.86 0.09 4.73 3812 pZS*13S-1916 3.82 0.08 4.89 3812pZS*13S-1917 3.61 0.08 2.70 3812 pZS*13S-1917 3.99 0.06 2.92 3812pZS*13S-1917 3.59 0.12 2.74 3812 pZS*13S-1917 3.57 0.11 2.64 3812pZS*13S-1918 3.10 0.25 6.43 3812 pZS*13S-1918 2.93 0.34 5.94 3812pZS*13S-1918 2.98 0.23 6.48 3812 pZS*13S-1918 3.03 0.23 6.53 3812pZS*13S-1920 3.30 11.20 44.10 3812 pZS*13S-1920 3.57 16.60 64.20 3812pZS*13S-1920 3.71 15.00 59.10 3812 pZS*13S-1920 3.55 16.30 62.40 3812pZS*13S-1921 3.99 0.23 9.07 3812 pZS*13S-1921 3.70 0.24 8.76 3812pZS*13S-1921 4.01 0.24 9.79 3812 pZS*13S-1921 3.88 0.27 9.35 3812pZS*13S-1922 2.46 0.06 4.00 3812 pZS*13S-1922 2.79 0.23 5.55 3812pZS*13S-1922 2.61 0.23 5.25 3812 pZS*13S-1922 2.93 0.18 5.53 3812pZS*13S-1923 2.82 0.25 6.46 3812 pZS*13S-1923 2.48 0.22 6.43 3812pZS*13S-1923 2.69 0.17 4.42 3812 pZS*13S-1923 3.18 0.18 10.50

EXAMPLE XXVI Production of 4HB and BDO Upon Expression of Genes EncodingCoA-dependant Succinate Semialdehyde Dehydrogenase Enzymes

This example describes production of 4HB and BDO upon expression ofgenes encoding CoA-dependant succinate semialdehyde dehydrogenaseenzymes.

Several heterologous CoA-dependant succinate semialdehyde dehydrogenasegenes (Table 38) were expressed in E. coli from the pZS*-13S plasmid toallow a functional pathway from succinyl-CoA to 4-hydroxybutyrate and1,4-butanediol. Host strain 3744 is based on strain ECKh-432 whoseconstruction is described above and in US20110045575. Notablemodifications include deletions in adhE, ldhA, pflB, mdh, and arcA.Strain ECKh-432 also contains chromosomally integrated copies of sucDfrom Porphyromonas gingivalis (encoding CoA-dependant succinatesemialdehyde dehydrogenase), sucA from Mycobacterium bovis (encodingalpha-ketoglutarate decarboxylase), 4hbd from P. gingivalis (encoding4-hydroxybutyrate dehydrogenase), and 4hbd from Clostridium kluyveri(encoding 4-hydroxybutyrate dehydrogenase). For this study, the M. bovissucA and P. gingivalis sucD genes were deleted in strain 3744.Additionally, strain 3744 contains a chromosomally integrated copy of4-hydroxybutyryl-CoA transferase expressed from the pA promoter at theattB locus. It also contains a pPSX23R plasmid expressing4-hydroxybutyryl-CoA reductase and 4-hydroxybutyraldehyde reductase.Table 39 shows that expression of heterologous CoA-dependant succinatesemialdehyde dehydrogenase genes enhances 4hb and/or BDO production instrain 3744 over the controls denoted by “pZS*13S”. Cells were culturedusing the 96 well-plate protocol described previously.

TABLE 38 Genes Encoding Functional CoA-dependant Succinate SemialdehydeDehydrogenase Enzymes Gene Number Organism Accession GI 35 Porphyromonasgingivalis NP_904963.1 34540484 W83 49 Clostridium difficile 630YP_001088857.1 126699960 1870 Metallosphaera sedula YP_001190808.1146303492 1871 Porphyromonas endodontalis ZP_04389701.1 229495977 ATCC35406 1872 Clostridium sporogenes ZP_02995751.1 187779278 1931Porphyromonas ZP_07821123.1 313887434 asaccharolytica DSM 20707 1932Odoribacter splanchnicus YP_004253242.1 325280700 DSM 20712

TABLE 39 BDO and 4-hydroxybutyrate (4HB) production of E. coli hoststrain 3744 containing several alternative CoA-dependant succinatesemialdehyde dehydrogenase plasmid constructs. The plasmid constructdenoted by only “pZS*13S” contains no CoA-dependant succinatesemialdehyde dehydrogenase gene. Plasmid constructs denoted by pZS*13Sfollowed by a number express the CoA-dependant succinate semialdehydedehydrogenase gene denoted by such number. BDO and 4HB concentrationsare in mM. LCMS - 16 h LCMS - 16 h Host strain Plasmid OD - 16 h 4HB BDO3744 pZS*13S 3.10 0.47 3.08 3744 pZS*13S 2.97 0.54 2.69 3744 pZS*13S3.18 0.89 4.09 3744 pZS*13S 1.26 0.44 2.41 3744 pZS*13S-035 3.63 7.2930.80 3744 pZS*13S-035 2.89 6.86 28.30 3744 pZS*13S-035 3.22 6.64 28.403744 pZS*13S-035 3.73 7.55 28.80 3744 pZS*13S-049 1.87 10.50 23.30 3744pZS*13S-049 1.65 10.10 23.70 3744 pZS*13S-049 1.87 10.80 23.60 3744pZS*13S-049 1.75 11.40 23.70 3744 pZS*13S-1870 3.58 0.43 7.18 3744pZS*13S-1870 3.51 0.57 7.28 3744 pZS*13S-1870 3.15 0.50 5.59 3744pZS*13S-1870 2.98 0.44 6.78 3744 pZS*13S-1871 3.33 6.73 37.20 3744pZS*13S-1871 3.28 6.42 31.90 3744 pZS*13S-1871 3.20 7.18 32.40 3744pZS*13S-1871 3.44 7.03 33.90 3744 pZS*13S-1872 3.52 0.44 4.52 3744pZS*13S-1872 3.54 0.49 4.71 3744 pZS*13S-1872 3.56 0.38 4.51 3744pZS*13S-1872 3.37 0.37 4.49 3744 pZS*13S-1872 3.47 0.50 4.32 3744pZS*13S-1872 3.58 0.56 4.68 3744 pZS*13S-1872 3.47 0.49 4.27 3744pZS*13S-1872 3.90 0.43 4.51 OD - 24 hr LCMS - 24 h LCMS - 24 h 3744pZS*13S 3.89 0.12 8.85 3744 pZS*13S 3.79 0.17 8.57 3744 pZS*13S 3.850.17 8.87 3744 pZS*13S 3.84 0.12 9.05 3744 pZS*13S-1931 3.78 3.18 45.103744 pZS*13S-1931 3.64 3.28 46.50 3744 pZS*13S-1931 2.91 4.25 34.00 3744pZS*13S-1931 3.73 3.22 45.40 3744 pZS*13S-1932 3.71 3.10 84.20 3744pZS*13S-1932 3.63 3.24 79.50 3744 pZS*13S-1932 3.61 3.18 82.60 3744pZS*13S-1932 3.61 3.24 83.90

EXAMPLE XXVII Production of 4HB and BDO Upon Expression of GenesEncoding 4-hydroxybutyrate Dehydrogenase Enzymes

This example describes production of 4HB and BDO upon expression ofgenes encoding 4-hydroxybutyrate dehydrogenase enzymes.

Several heterologous 4-hydroxybutyrate dehydrogenase genes (Table 40)were expressed in E. coli from the pZS*-13S plasmid to allow afunctional pathway from succinyl-CoA and alpha-ketoglutarate to4-hydroxybutyrate and 1,4-butanediol. Host strain 3891 is based onstrain ECKh-432 whose construction is described above and inUS20110045575. Notable modifications include deletions in adhE, ldhA,pflB, mdh, and arcA. Strain ECKh-432 also contains chromosomallyintegrated copies of sucD from Porphyromonas gingivalis (encodingCoA-dependant succinate semialdehyde dehydrogenase), sucA fromMycobacterium bovis (encoding alpha-ketoglutarate decarboxylase), 4hbdfrom P. gingivalis (encoding 4-hydroxybutyrate dehydrogenase), and 4hbdfrom Clostridium kluyveri (encoding 4-hydroxybutyrate dehydrogenase).For this study, the P. gingivalis and C. kluyveri 4hbd genes weredeleted in strain 3891. Additionally, strain 3891 contains achromosomally integrated copy of 4-hydroxybutyryl-CoA transferaseexpressed from the pA promoter at the attB locus. It also contains apPSX23R plasmid expressing 4-hydroxybutyryl-CoA reductase and4-hydroxybutyraldehyde reductase. Table 41 shows that expression ofheterologous 4-hydroxybutyrate dehydrogenase genes enhances 4hb and/orBDO production in strain 3891 over the controls denoted by “pZS*13S”.Cells were cultured using the 96 well-plate protocol describedpreviously.

TABLE 40 Genes encoding functional 4-hydroxybutyrate dehydrogenaseenzymes. Gene Number Organism Accession GI 36 Porphyromonas gingivalisNP_904964.1 34540485 W83 150 Clostridium acetobutylicum NP_348201.115894852 ATCC 824 1879 Porphyromonas endodontalis ZP_04389726.1229496002 ATCC 35406 1880 Porphyromonas YP_004441566.1 332299645asaccharolytica DSM 20707 1881 Odoribacter splanchnicus YP_004253243.1325280701 DSM 20712 1882 Eubacterium saphenum ZP_05427218.1 255994083ATCC 49989 1883 Clostridium ljungdahlii YP_003782020.1 300857036 DSM13528 1884 Clostridium perfringens YP_694972.1 110800333 ATCC 13124 1888Clostridium difficile 630 YP_001088853.1 126699956 1889 Geobactersulferreducens NP_952425.1 39996474 1894 Metallosphaera sedulaYP_001191506.1 146304190 DSM 5348 (ATCC 51363D-5)

TABLE 41 BDO and 4-hydroxybutyrate (4HB) production of E. coli hoststrain 3891 containing several alternative 4-hydroxybutyratedehydrogenase plasmid constructs. The plasmid construct denoted by only“pZS*13S” contains no 4-hydroxybutyrate dehydrogenase gene. Plasmidconstructs denoted by pZS*13S followed by a number express the4-hydroxybutyrate dehydrogenase gene denoted by such number. BDO and 4HBconcentrations are in mM. LCMS - 16 h LCMS - 16 h Host Plasmid OD - 16 h4HB BDO 3891 pZS*13S 3.26 0.07 1.33 3891 pZS*13S 3.06 0.04 1.38 3891pZS*13S 3.17 0.04 1.80 3891 pZS*13S 3.22 0.04 1.25 3891 pZS*13S-036 3.117.19 15.90 3891 pZS*13S-036 2.95 7.31 17.40 3891 pZS*13S-036 3.17 7.0016.40 3891 pZS*13S-036 2.80 6.84 16.60 3891 pZS*13S-150 2.99 2.58 31.203891 pZS*13S-150 3.41 2.49 33.30 3891 pZS*13S-150 3.11 2.88 32.40 3891pZS*13S-150 2.91 2.71 33.30 3891 pZS*13S-1879 3.35 1.99 9.78 3891pZS*13S-1879 3.12 2.26 10.80 3891 pZS*13S-1879 3.42 2.06 9.58 3891pZS*13S-1879 3.16 2.21 10.20 3891 pZS*13S-1880 2.01 3.99 13.30 3891pZS*13S-1880 2.85 4.49 14.00 3891 pZS*13S-1880 2.99 4.52 12.90 3891pZS*13S-1880 2.95 4.04 12.70 3891 pZS*13S-1881 3.18 3.73 7.00 3891pZS*13S-1881 2.95 3.56 7.64 3891 pZS*13S-1881 3.03 3.40 6.98 3891pZS*13S-1881 2.96 3.68 7.50 3891 pZS*13S-1882 3.20 3.62 22.70 3891pZS*13S-1882 3.13 3.85 24.10 3891 pZS*13S-1882 3.33 3.34 22.20 3891pZS*13S-1882 2.60 3.67 19.80 3891 pZS*13S-1883 3.53 2.47 24.00 3891pZS*13S-1883 3.39 1.79 24.60 3891 pZS*13S-1883 3.41 2.20 27.10 3891pZS*13S-1883 3.33 2.13 22.00 3891 pZS*13S-1884 3.09 1.13 30.50 3891pZS*13S-1884 3.02 1.20 33.80 3891 pZS*13S-1884 3.76 1.34 34.40 3891pZS*13S-1884 3.15 1.40 36.80 3891 pZS*13S-1888 3.11 0.28 31.40 3891pZS*13S-1888 3.04 0.22 30.70 3891 pZS*13S-1888 3.09 0.21 29.90 3891pZS*13S-1888 3.33 0.30 31.50 3891 pZS*13S-1889 3.17 1.03 19.20 3891pZS*13S-1889 3.04 1.12 18.70 3891 pZS*13S-1889 3.14 1.05 17.40 3891pZS*13S-1889 3.01 1.25 18.10 3891 pZS*13S-1894 3.03 0.85 13.70 3891pZS*13S-1894 2.91 0.67 13.00 3891 pZS*13S-1894 3.00 1.04 12.80 3891pZS*13S-1894 3.03 0.86 12.90

EXAMPLE XXVIII Production of 4HB and BDO Upon Expression of GenesEncoding 4-Hydroxybutyrate Transferase Enzymes

This example describes production of 4hb and BDO upon expression ofgenes encoding 4-hydroxybutyrate transferase enzymes.

Several heterologous 4-hydroxybutyrate transferase genes (Table 42) wereexpressed in E. coli from either the pZS*-13S or F′ plasmid to allow afunctional pathway from alpha-ketoglutarate and succinyl-CoA to1,4-butanediol. Host strains include 1269, 2731 Δcat2, and 1654. Thesehost strains are based on strain ECKh-432 whose construction isdescribed above and in US20110045575. Notable modifications includedeletions in adhE, ldhA, pflB, mdh, and arcA. Strain ECKh-432 alsocontains chromosomally integrated copies of sucD from Porphyromonasgingivalis (encoding CoA-dependant succinate semialdehydedehydrogenase), sucA from Mycobacterium bovis (encodingalpha-ketoglutarate decarboxylase), 4hbd from P. gingivalis (encoding4-hydroxybutyrate dehydrogenase), and 4hbd from Clostridium kluyveri(encoding 4-hydroxybutyrate dehydrogenase). Strains 1269, 2731 Δcat2,and 1654 were all tested with 4-hydroxybutyryl-CoA reductase and4-hydroxybutyraldehyde reductase expressed from the pZS*-13S plasmid.Tables 43, 44, and 45 show that expression of heterologous4-hydroxybutyrate transferase genes allows BDO production in strains1269, 2731 Δcat2, and 1654, respectively. Cells were cultured using the96 well-plate protocol described previously.

TABLE 42 Genes encoding functional 4-hydroxybutyrate transferaseenzymes. Gene Number Organism Accession GI  34 Porphyromonas gingivalisNP_904965 34540486 W83 1210 Clostridium difficile 630 YP_001088854126699957 1769 Eubacterium saphenum ZP_05427217 255994082 ATCC 499891772 Porphyromonas endodontalis ZP_04389695 229495971 ATCC 35406 1774Anaerostipes caccae ZP_02417601 167745474 DSM 14662 33B Clostridiumaminobutyricum CAB60036 188032706 1801 Fusobacterium nucleatumZP_00144049 34763077 subsp. vincentii ATCC 49256 1803 Fusobacteriumulcerans ZP_07928926 317064441 ATCC 49185 1804 Fusobacterium variumZP_08693772 340757169 ATCC 27725 1805 Odoribacter splanchnicusYP_004253244 325280702 DSM 20712 1807 Porphyromonas gingivalisYP_001930004 188995752 1808 Fusobacterium ZP_07914775 315918535gonidiaformans ATCC 25563 1809 Acetonema longum ZP_08623452 338811223DSM 6540

The nucleotide sequences for 1210C and 033B are provided below as thesewere codon-optimized for expression in E. coli.

33B: (SEQ ID NO: 174)atggactggaagaagatctatgaagacagaacctgcactgcagatgaagcagtaaagagcattaagtcaggtgaccgcgtgctgtttgcgcactgtgttgctgaaccgccagttctggtagaagcaatggttgcgaatgcagctgcatacaagaatgtcacggtttcacacatggttacccttggtaagggtgaatactcaaaaccagaatataaagaaaactttacttttgaaggttggtttaccagcccttcaacacgtggatccattgcagaaggacacggacagtttgtccctgtattcttccacgaggtaccatctttaatccgtaaagacattttccatgttgatgtattcatggtaatggtatccccgccagatcataacggtttctgctgtgtgggtgtatcttctgactataccatgcaggctatcaaatcagcaaaaattgtactggctgaagtgaatgatcaggtacctgtagtttatggcgatacctttgttcacgttagtgaaatcgacaagttcgttgaaacttcacatccactgccagaaatcggtctgccgaagatcggtgaagtagaagctgctattggtaagcactgcgcttcgctgatcgaagatggttccacattacagcttggtatcggcgctattccggatgctgtactttcacagcttaaggacaagaaacaccttggtatccactctgaaatgatttccgacggtgttgttgatctttacgaagcaggcgttattgactgcagccaaaagtctatcgacaaaggcaaaatggcaataacattcttaatgggaacgaagcgtctttatgatttcgctgcaaacaatccaaaggttgaattaaagccggttgactacatcaatcatccatctgtagttgcacagtgcagcaaaatggtttgcatcaatgcttgcttgcaagttgattttatgggtcagattgtctccgatagtattggcacaaagcagttctccggcgtaggcggtcaggttgacttcgtacgcggtgcatccatgtctattgacggcaaaggtaaagcgatcatcgcgatgccttccgttgcaaagaagaaagatggcagtatgatttcgaagatcgttccattcatcgatcacggtgcagctgtaactacatcccgtaacgatgcggactatgtcgtaacggaatatggtattgctgaaatgaagggtaagtcgttacaggaccgcgcacgcgcgttaatcaatattgcccaccctgatttcaaagatgaattaaaggctgaatttgaaaagcgcttcaacgcggcattctaa. 1210C:(SEQ ID NO: 175)atgagctggcaagaactgtatcaaagtaaattatgttcagccacagaagcggtaaaacagattaaaaacggtgataccgtggtatttgcccattgtgtaggtgaaccgcctgcactggtggaggcgatgattgaaaatgctgaacaatataaagatgttgagattaaacatatggttagcctgggtagtggtggttatactgcgaaagggatggaagcgcattttcgcgtaaatccaatgtttgtcagcggcaatgtacgtaaggcgattgaaaatggcgatggtgattttacacctgcattcttccatgaagtaccaaagttgctgcgtgaaaaacgtctgaaatgtgatgttgttctggcacaggtaacgccaccagatgaacatggttattgttcgctgggaacaagcgttgattatacctatgaagccattaaaaccgcgcgcaccgtaattgttcaggtgaatgaccagtttcctcgcacctatggtgaggtggtgcatgtcagcgagtttgactatatcgttgaaaaatcacaaccgctgtttgaactgcaacctgcaaagattggcgaagttgaagaagcgattggtaaaaattgtgcctcgctgattgaagatggtagcacgttacagctggggattggtgggattccggatgcggtgatgttatttctgactgataaaaaagatttagggattcatagcgaaatgattagcgatggcacgctggcgctttatgaaaaaggtgttattaatggtaaatataaaaattttgataaagaaaaaatgacggttaccttcctgatgggtactaaaaaactgtatgactttgccaataataacccggcagtagaggtaaaaccggtagactatgtgaatcatccggcaattatcatgaaacaacataagatggtttctattaatagcgccattcaggttgatttaatggggcaggtggttgcagaggcgatgggactgcgccaattttccggtgttggcggtcaggttgactttattcgtggcgtgtcgatgggtgaagatggcaaggcgattatcgcgatgccttcaatcactacaaaaaaagatggtacggtaattagcaaaatcgtctctattgtcgatgaaggtgcaccgattaccacctcacgtaatgatgttgattatattgtcacagaatacggtattgcagaattaaaaggcaaatcgctgcgtgaacgcgcacgtaatctgattaatattgctcatccatcggtacgtgaatcgctggcagtagaatttgaaaagcgctttaaagagaaatattaa.

TABLE 43 BDO, GBL, and 4-hydroxybutyrate (4HB) production of E. colihost strain 1269 containing two alternative 4-hydroxybutyratetransferase plasmid constructs. The plasmid construct denoted by only“pZA33S (empty)” contains no 4-hydroxybutyrate transferase gene. Plasmidconstructs denoted by F′ followed by a number express the4-hydroxybutyrate transferase gene denoted by such number. BDO and 4HBconcentrations are in mM. LCMS- LCMS- LCMS- 24 h 24 h 24 h Host StrainPlasmid OD 24 h 4HB BDO GBL 1269 pZA33S 2.92 27.60 0.00 0.00 (empty)1269 pZA33S 2.12 23.80 0.59 0.07 (empty) 1269 pZA33S 2.72 28.80 2.130.03 (empty) 1269 pZA33S 2.32 30.50 0.14 0.00 (empty) 1269 F′34 1.762.42 22.20 0.87 1269 F′34 1.70 2.34 21.00 0.88 1269 F′34 2.09 3.23 23.501.21 1269 F′34 3.83 2.77 51.30 5.01 1269 F′1210C 1.99 2.84 28.20 1.731269 F′1210C 1.64 2.73 27.30 1.61 1269 F′1210C 1.89 4.28 32.30 2.59 1269F′1210C 3.47 3.76 56.38 7.22

TABLE 44 BDO, GBL, and 4-hydroxybutyrate (4HB) production of E. colihost strain 2731 Δcat2 containing alternative 4-hydroxybutyratetransferase plasmid constructs. Plasmid constructs denoted bypZS*13S-followed by a number express the 4-hydroxybutyrate transferasegene denoted by such number. The pZS*13S-backbone also expressesfunctional 4-hydroxybutyryl-CoA reductase and 4-hydroxybutyraldehydedehydrogenase genes. BDO and 4HB concentrations are in mM. LCMS- LCMS-LCMS- 19 h 19 h 19 h Host Strain Plasmid OD 19 h 4HB BDO GBL 2731 Δcat2pZS*13S-1769 3.75 1.1 24.9 0.1 2731 Δcat2 pZS*13S-1769 3.84 1.3 23.9 0.12731 Δcat2 pZS*13S-1769 4.10 1.3 23.0 0.1 2731 Δcat2 pZS*13S-1769 4.141.1 27.3 0.1 2731 Δcat2 pZS*13S-1772 4.33 2.9 36.7 0.2 2731 Δcat2pZS*13S-1772 4.37 2.3 37.9 0.3 2731 Δcat2 pZS*13S-1772 4.02 2.7 40.4 0.32731 Δcat2 pZS*13S-1772 3.59 3.4 27.4 0.2 2731 Δcat2 pZS*13S-1774 4.102.2 29.9 0.2 2731 Δcat2 pZS*13S-1774 3.93 1.9 29.2 0.3 2731 Δcat2pZS*13S-1774 4.26 2.5 31.5 0.3 2731 Δcat2 pZS*13S-1774 4.76 1.6 29.8 0.2

TABLE 45 BDO, GBL, and 4-hydroxybutyrate (4HB) production of E. colihost strain 1654 containing alternative 4-hydroxybutyrate transferaseplasmid constructs. Plasmid constructs denoted by pZS*13S- followed by anumber express the 4-hydroxybutyrate transferase gene denoted by suchnumber. The pZS*13S- backbone also expresses functional4-hydroxybutyryl- CoA reductase and 4-hydroxybutyraldehyde dehydrogenasegenes. BDO and 4HB concentrations are in mM. LCMS- LCMS- LCMS- 19 h 19 h19 h Host Strain Plasmid OD 19 h 4HB BDO GBL 1654 pZS*-13S-033B 3.961.47 26.00 1.69 1654 pZS*-13S-033B 4.17 1.32 23.90 1.54 1654pZS*-13S-033B 4.11 0.99 25.80 1.62 1654 pZS*-13S-033B 3.80 1.04 25.401.61 1654 pZS*-13S-1801 3.68 1.88 21.80 1.61 1654 pZS*-13S-1801 3.901.35 21.70 1.67 1654 pZS*-13S-1801 3.76 1.76 20.90 1.55 1654pZS*-13S-1801 3.66 1.92 22.00 1.77 1654 pZS*-13S-1803 3.69 1.71 27.601.80 1654 pZS*-13S-1803 3.79 1.45 23.60 1.63 1654 pZS*-13S-1803 3.641.09 24.80 1.72 1654 pZS*-13S-1803 3.64 1.30 23.60 1.67 1654pZS*-13S-1804 3.74 1.27 26.00 1.83 1654 pZS*-13S-1804 3.87 1.90 25.401.95 1654 pZS*-13S-1804 4.04 1.48 22.80 1.65 1654 pZS*-13S-1804 3.751.87 26.00 1.87 1654 pZS*-13S-1805 3.84 1.47 25.20 1.47 1654pZS*-13S-1805 3.75 1.06 27.50 1.54 1654 pZS*-13S-1805 3.08 1.67 22.001.26 1654 pZS*-13S-1805 3.57 1.22 23.80 1.36 1654 pZS*-13S-1807 3.240.00 4.80 0.40 1654 pZS*-13S-1807 3.30 0.29 5.10 0.48 1654 pZS*-13S-18073.36 0.17 4.68 0.39 1654 pZS*-13S-1807 3.31 0.21 5.16 0.38 1654pZS*-13S-1808 3.38 2.39 24.50 1.20 1654 pZS*-13S-1808 3.39 2.56 22.201.07 1654 pZS*-13S-1808 3.45 2.16 22.40 1.10 1654 pZS*-13S-1808 3.422.26 23.10 1.27 1654 pZS*-13S-1809 4.26 1.52 18.10 0.93 1654pZS*-13S-1809 4.37 1.86 21.50 1.07 1654 pZS*-13S-1809 3.51 1.67 16.000.81 1654 pZS*-13S-1809 4.49 0.97 18.60 0.97

EXAMPLE XXIX Production of 4HB and BDO Upon Expression of Genes Encoding4-Hydroxybutyryl-CoA Reductase Enzymes

This example describes production of 4hb and BDO upon expression ofgenes encoding 4-hydroxybutyryl-CoA reductase enzymes.

Several heterologous 4-hydroxybutyryl-CoA reductase genes (Table 46)were expressed in E. coli from the pZS*-13S plasmid to allow afunctional pathway from succinyl-CoA and alpha-ketoglutarate to1,4-butanediol. Host strain 1889 is based on strain ECKh-432 whoseconstruction is described above and in US20110045575. Notablemodifications include deletions in adhE, ldhA, pflB, mdh, and arcA.Strain ECKh-432 also contains chromosomally integrated copies of sucDfrom Porphyromonas gingivalis (encoding CoA-dependant succinatesemialdehyde dehydrogenase), sucA from Mycobacterium bovis (encodingalpha-ketoglutarate decarboxylase), 4hbd from P. gingivalis (encoding4-hydroxybutyrate dehydrogenase), and 4hbd from Clostridium kluyveri(encoding 4-hydroxybutyrate dehydrogenase). Strain 1889 containsdeletions in the native alcohol dehydrogenase genes adhP, yqhD, yahK,and yjgB. Additionally, strain 1889 contains a chromosomally integratedcopy of 4-hydroxybutyryl-CoA transferase expressed from the pA promoterat the attB locus. Strain 1889 contains the F′ plasmid expressing afunctional 4-hydroxybutyraldehyde reductase gene. Strain 4269 contains achromosomally integrated copy of a 4-hydroxybutyraldehyde reductase geneat the hemN locus under control of the p119 promoter. Tables 47 and 48show that expression of heterologous 4-hydroxybutyryl-CoA reductasegenes enhances BDO production in strains 1889 and 4269, respectively,over the controls denoted by “pZS*13S”. Cells were cultured using the 96well-plate protocol described previously.

TABLE 46 Genes encoding functional 4-hydroxybutyryl-CoA reductaseenzymes. Gene Number Organism Accession GI  025B Clostridiumbeijerinckii NCIMB YP_001310903.1 150018649 8052  733 Clostridiumhylemonae DSM ZP_03778292.1 225569267   15053  744 Clostridiummethylpentosum ZP_03705305.1 225016072   DSM 5476  787 Eubacteriumhallii DSM 3353 ZP_03715465.1 225026273  865 Ruminococcus obeum ATCCZP_01962381.1 153809713   29174  778 Bacillus selenitireducens MLS10YP_003701164.1 297585384  714 Clostridium AAP42563.1 31075383  saccharoperbutylacetonicum   N1-4  707 Lactobacillus brevis ATCC 367YP_795711.1 116334184  141 Desulfatibacillum alkenivorans YP_002434126.1218782808   AK-01  133 Clostridium phytofermentans YP_001558295.1160879327   ISDg  715 Clostridium bolteae ATCC ZP_02089671.1 160942363  BAA-613  779 Photobacterium profundum ZP_01222600.1 90414628   3TCK  145Citrobacter koseri ATCC BAA- YP_001452373.1 157145054   895  704Salmonella enterica typhimurium NP_460996.1 16765381  777 Sebaldellatermitidis ATCC YP_003307836.1 269119659   33386  795 Fusobacteriumnucleatum subsp. ZP_04969437.1 254302079   polymorphum ATCC 10953  706Tolumonas auensis DSM 9187 YP_002892893.1 237808453  137 Rhodospirillumrubrum ATCC YP_426002.1 83592250 11170 1993 Metalibrary sp N/A N/A

TABLE 47 BDO and 4-hydroxybutyrate (4HB) production of E. coli hoststrain 1889 containing several alternative 4-hydroxybutyryl-CoAreductase plasmid constructs. The plasmid construct denoted by only“pZS*13S” contains no 4-hydroxybutyryl-CoA reductase gene. Plasmidconstructs denoted by pZS*13S followed by a number express the4-hydroxybutyryl-CoA reductase gene denoted by such number. BDO and 4HBconcentrations are in mM. LCMS - 24 hrs LCMS - 24 hrs Host Plasmid OD 24h 4HB BDO 1889 pZS*13S 3.12 3.93 0 1889 pZS*13S 3.68 10.4 0.5 1889pZS*13S 2.81 2.74 0 1889 pZS*13S 2.61 1.16 0 1889 pZS*13S-025B 3.25 0.878.42 1889 pZS*13S-025B 2.11 0.8 7.09 1889 pZS*13S-025B 3.01 0.65 7.381889 pZS*13S-025B 3.04 0.72 6.81 1889 pZS*13S-733 1.67 1.07 1.64 1889pZS*13S-733 1.63 1.22 1.87 1889 pZS*13S-733 2.16 1.16 1.89 1889pZS*13S-733 2.34 1.04 1.88 1889 pZS*13S-744 2.75 0.63 2.17 1889pZS*13S-744 3.00 0.65 1.75 1889 pZS*13S-744 2.75 0.5 1.72 1889pZS*13S-744 1.98 0.39 0.56 1889 pZS*13S-787 2.63 0.88 1.82 1889pZS*13S-787 2.40 1 1.75 1889 pZS*13S-787 2.88 1.06 2.41 1889 pZS*13S-7873.26 1.03 1.86 1889 pZS*13S-865 2.60 0.95 1.71 1889 pZS*13S-865 2.701.11 2.09 1889 pZS*13S-865 2.69 0.98 1.4 1889 pZS*13S-865 2.70 1.06 1.741889 pZS*13S-778 2.76 1.15 5.79 1889 pZS*13S-778 3.12 1.06 6.48 1889pZS*13S-778 2.83 1.08 4.98 1889 pZS*13S-778 2.79 1.1 5.19 1889pZS*13S-714 2.60 0.63 6.55 1889 pZS*13S-714 2.77 0.86 6.88 1889pZS*13S-714 2.67 0.77 6.51 1889 pZS*13S-714 2.73 0.75 8.12 1889pZS*13S-707 1.39 0.3 1.56 1889 pZS*13S-707 1.05 0.24 1.57 1889pZS*13S-707 1.99 0.26 1.62 1889 pZS*13S-707 2.20 0.28 1.76 1889pZS*13S-141 2.54 1.08 2.13 1889 pZS*13S-141 2.72 1 2.01 1889 pZS*13S-1412.54 1.04 1.9 1889 pZS*13S-141 2.34 0.97 1.77 1889 pZS*13S-133 2.63 0.72.69 1889 pZS*13S-133 1.79 0.73 2.5 1889 pZS*13S-133 3.04 0.69 2.26 1889pZS*13S-133 3.21 0.65 2.84 1889 pZS*13S-715 2.79 1.03 2.31 1889pZS*13S-715 2.67 1.13 1.55 1889 pZS*13S-715 2.90 1.02 1.6 1889pZS*13S-715 3.10 1.17 2.26 1889 pZS*13S-779 3.21 8.88 0.46 1889pZS*13S-779 2.84 10.5 0.63 1889 pZS*13S-779 3.98 11.9 0.84 1889pZS*13S-779 4.55 13.5 1.15 1889 pZS*13S-145 2.08 0.53 0.76 1889pZS*13S-145 2.65 0.66 0.72 1889 pZS*13S-145 2.74 0.55 0.63 1889pZS*13S-145 3.08 0.7 0.68 1889 pZS*13S-704 2.49 0.93 1.65 1889pZS*13S-704 2.08 0.89 1.81 1889 pZS*13S-704 2.97 0.76 1.58 1889pZS*13S-704 3.35 0.9 2.32 1889 pZS*13S-777 2.65 1.45 0.94 1889pZS*13S-777 3.03 1.89 1.5 1889 pZS*13S-777 3.36 2.16 1.79 1889pZS*13S-777 3.10 1.93 1.6 1889 pZS*13S-795 2.60 3.22 1.55 1889pZS*13S-795 3.43 4.54 1.96 1889 pZS*13S-795 3.33 4.24 1.93 1889pZS*13S-795 2.87 2.15 0.97 1889 pZS*13S-706 2.67 0.96 1.36 1889pZS*13S-706 2.41 1.52 2.64 1889 pZS*13S-706 2.94 1.4 2.56 1889pZS*13S-706 2.71 0.85 0.89 1889 pZS*13S-137 2.85 1.76 1.4 1889pZS*13S-137 3.04 2.31 2.21 1889 pZS*13S-137 3.00 1.89 1.56 1889pZS*13S-137 2.45 0.55 0.17

TABLE 48BDO and 4-hydroxybutyrate (4HB) production of E. coli host strain 4269 containing eitherno heterologous 4-hydroxybutyryl-CoA reductase gene (i.e., pZS*13S) or expressing the 4-hydroxybutyryl-CoA reductase gene, 1993, from the pZS* plasmid under control of the p100promoter. The gene sequence for 1993 is provided in the bottom column of the table. BDO and4HB concentrations are in mM. LCMS- LCMS- 24 hrs 24 hrs Host PlasmidOD 24 h 4HB BDO 4269 pZS*13S 3.12 49.3 1.9 4269 pZS*13S 3.08 50.7 1.74269 pZS*13S 3.31 48.2 3.0 4269 pZS*13S 3.18 55.7 3.2 4269pZS*13S-p100-1993 3.88 13.1 37.2 4269 pZS*13S-p100-1993 3.58 18.3 44.34269 pZS*13S-p100-1993 3.40 17.3 46.9 4269 pZS*13S-p100-1993 3.40 16.046.5 Gene sequence for 1993: (SEQ ID NO: 176)atggatttgaagctgaccgatgcggacgttgaggcaatcgtagcgcaagtcatggctaacattgagcgccgtctgggtagcgcggaagcgggcagcgcagcatccgctgcgtcgccggcaccggctgcgccggttcgtaccgctccgggtggcagcccggcagccagcccgcgtccggagtacggtgtttttgatcgtgcggaggatgcagtcgccgctgccgccgaagctcaggaagccttcctgcgccagtgtcgtctgcaagaccgtgagcgcattctgcgtgccatccgtgaagagactctggcacgcaaggaagaattggcacgcctgatttgggaggaaacgaagctgggtcgcttggaacacaaaattgcaaagctggaattgacggcgctgaaaaccccgggtacggaggatctgcgcaccgaggcatttagcggcgacaacggcctgaccatcgtcgaacatgcgccgtacggtgtgattggcgcggttaccccggttacgaaccctgcggagactattatcaacaacgcgatcggcatgctggcaagcggtaatgcagtggtgttcaacgtgcaccctagcgctaaacgttgctccgcgtataccgtccagatgatcaataaagcagtgatggcagcgggtggtccgccgaatctggttacgatggttcgcgagccgaccatggaaaccctgaatgcgatcatccgcagcccgagcgtgaagctgctggtgggcaccggcggtccgggtctggttagcaccctgctgcgctctggtaagaaagcgattggtgcgggtgcgggcaatccgccggtcgttgtggatgataccgccgacctggagcatgcggcaaaggaaatcatcaaaggcgcgtcctttgacaacaatattctgtgtattgcggagaaagaggtttttgtggttgataaagcggccgacggtctgatctatcacatgctggataacggcgcatacatgctgggtcgtgacgagctggagaaggtgatgcaattcgcgctgaccgcggacgaaagccagggcggtgcgggttgctctattgatccgcgtcgtgcgtggcatgtgactaaggagtgggtgggcaaagatgcgcgcttgttcctggagaagattggtgtcaagactgaccgtccggttgacttgctgttgtgcgaggttgactttgaccacccgttcgtgcagctggaacaaatgatgccagtactgccgattgttcgtgtccgtgacctggatgaagccatcggtatggccgtccgtgcggagcacggcaatcgtcacaccgcaattatgcatagccgtaacgtggacaatctgacccgttttgcccgtgccattgccacgaccatcttcgtcaaaaacgcaagcagcttggcgggtgttggttatggcggtgaaggttttaccaccatgacgatcgccggtccaacgggcgagggtctgacgtcggctcgtacgttcacgcgcaaagtccgctgtgtcctggcggacggcggtttccgtatcgttggttaa

EXAMPLE XXX Production of 4HB and BDO Upon Expression of Genes Encoding4-Hydroxybutyraldehyde Reductase Enzymes

This example describes production of 4hb and BDO upon expression ofgenes encoding 4-hydroxybutyraldehyde reductase enzymes.

Several heterologous 4-hydroxybutyraldehyde reductase genes (Table 49)were expressed in E. coli from the pZS*-13S plasmid to allow afunctional pathway from succinyl-CoA and alpha-ketoglutarate to1,4-butanediol. Host strain 1889 is based on strain ECKh-432 whoseconstruction is described above and in US20110045575. Notablemodifications include deletions in adhE, ldhA, pflB, mdh, and arcA.Strain ECKh-432 also contains chromosomally integrated copies of sucDfrom Porphyromonas gingivalis (encoding CoA-dependant succinatesemialdehyde dehydrogenase), sucA from Mycobacterium bovis (encodingalpha-ketoglutarate decarboxylase), 4hbd from P. gingivalis (encoding4-hydroxybutyrate dehydrogenase), and 4hbd from Clostridium kluyveri(encoding 4-hydroxybutyrate dehydrogenase). Strain 1889 containsdeletions in the native alcohol dehydrogenase genes adhP, yqhD, yahK,and yjgB. Additionally, strain 1889 contains a chromosomally integratedcopy of 4-hydroxybutyryl-CoA transferase expressed from the pA promoterat the attB locus. Table 50 shows that expression of heterologous4-hydroxybutyraldehyde reductase genes enhances BDO production in strain1889 over the controls denoted by “pZS*13S”. Cells were cultured usingthe 96 well-plate protocol described previously.

TABLE 49 Genes encoding functional 4-hydroxybutyraldehyde reductaseenzymes. Gene Number Organism Accession GI 12 Escherichia coli K-12MG1655 AAC76047.1 1789386 956 Clostridium beijerinckii NCIMB 8052YP_001309304.1 150017050 1247 Clostridium saccharobutylicum P13604.1113352 1248 Clostridium asparagiforme DSM 15981 ZP_03760651.1 2254054621250 Clostridium bolteae ATCC BAA-613 ZP_02083621.1 160936248 1251Clostridium cellulovorans 743B YP_003845251.1 302876618 1256 Clostridiumhiranonis DSM 13275 ZP_03294286.1 210624270 1259 Clostridiummethylpentosum DSM 5476 ZP_03705769.1 225016577 1364 Atopobium parvulumDSM 20469 YP_003179160.1 257783943 1365 Tolumonas auensis DSM 9187YP_002893476.1 237809036 1366 Clostridium carboxidivorans P7ZP_05394983.1 255528157

TABLE 50 BDO and 4-hydroxybutyrate (4HB) production of E. coli hoststrain 1889 containing several alternative 4-hydroxybutyraldehydereductase plasmid constructs. The plasmid construct denoted by only“pZS*13S-” contains no 4-hydroxybutyraldehyde reductase gene but doesexpress a 4-hydroxybutyryl-CoA reductase gene. Plasmid constructsdenoted by pZS*13S- followed by a number express both4-hydroxybutyryl-CoA reductase and the 4-hydroxybutyraldehyde reductasegene denoted by such number. BDO and 4HB concentrations are in mM.LCMS - 24 h LCMS - 24 h Host Plasmid OD 24 h 4HB BDO 1889 pZS*13S 4.6821.5 0.4 1889 pZS*13S 4.33 15.9 0.1 1889 pZS*13S 4.83 18.9 0.3 1889pZS*13S 4.25 19.8 0.6 1889 pZS*13S-956 4.45 2.4 52.0 1889 pZS*13S-9564.92 3.5 52.1 1889 pZS*13S-956 4.65 4.2 42.9 1889 pZS*13S-956 5.37 4.754.2 1889 pZS*13S-1247 4.99 2.4 46.6 1889 pZS*13S-1247 5.38 2.3 42.51889 pZS*13S-1247 5.68 2.1 38.6 1889 pZS*13S-1247 5.18 2.0 40.4 1889pZS*13S-1248 4.25 2.8 17.6 1889 pZS*13S-1248 4.4 1.9 17.8 1889pZS*13S-1248 3.06 2.1 17.3 1889 pZS*13S-1248 4.22 2.0 19.5 1889pZS*13S-1250 4.76 0.9 30.4 1889 pZS*13S-1250 5.05 1.0 30.9 1889pZS*13S-1250 4.67 1.1 28.5 1889 pZS*13S-1250 5.57 1.1 33.5 1889pZS*13S-1251 4.68 2.0 49.9 1889 pZS*13S-1251 4.65 2.0 45.3 1889pZS*13S-1251 4.86 1.7 42.6 1889 pZS*13S-1251 4.76 1.9 42.7 1889pZS*13S-1256 4.63 15.7 11.9 1889 pZS*13S-1256 4.48 16.2 12.6 1889pZS*13S-1256 5.1 15.0 12.8 1889 pZS*13S-1256 5.51 15.3 13.4 1889pZS*13S-1259 5.01 1.0 33.7 1889 pZS*13S-1259 5.29 1.1 28.9 1889pZS*13S-1259 5.49 1.3 29.5 1889 pZS*13S-1259 5.25 1.2 31.5 1889pZS*13S-1364 5.52 5.0 48.8 1889 pZS*13S-1364 5.12 8.3 47.7 1889pZS*13S-1364 5.47 7.1 42.2 1889 pZS*13S-1364 5.48 7.8 46.0 1889pZS*13S-1365 5.49 1.1 33.1 1889 pZS*13S-1365 5.65 0.7 37.7 1889pZS*13S-1365 6.08 1.1 34.8 1889 pZS*13S-1365 5.5 0.9 28.6 1889pZS*13S-1366 5.13 2.2 52.6 1889 pZS*13S-1366 4.76 2.4 56.1 1889pZS*13S-1366 4.93 2.2 56.9 1889 pZS*13S-1366 blank 2.5 57.9

In the examples below, additional genetic manipulations are describedthat were made in the host strain to improve the yield of BDO onglucose. The key genes for deletion and overexpression are shown in FIG.70.

Starting from the TCA cycle intermediate, alpha-ketoglutarate (AKG),there are two ways to channel flux into the BDO pathway. The first onecomprises of an α-ketoacid decarboxylase that decarboxylates AKG intosuccinate semialdehyde and subsequently reduces it to 4-hydroxybutyrate(4HB). An alternative way to get to 4HB is via AKG dehydrogenase whichtransforms AKG into succinyl-CoA. This is then reduced to succinatesemialdehye via succinate semialdehdye dehydrogenase and then to 4HB.4HB can then be activated to 4-hydroxybutyryl-CoA (4-HBCoA) via atransferase that transfers the CoA from acetyl-CoA. 4-HBCoA is reducedto 4-hydroxybutyraldehyde via an aldehyde dehydrogenase (ALD). Thealdehyde is finally reduced to 1,4-butanediol (BDO) using an alcoholdehydrogenase (ADH).

EXAMPLE XXXI Overexpression of Phosphoenolpyruvate Carboxylase

This example describes overexpression of phosphoenolpyruvate (PEP)carboxylase.

ppc refers to a gene that encodes for phosphoenolpyruvate (PEP)carboxylase activity. The net reaction involves the conversion of PEPand bicarbonate into oxaloacetate and phosphate. The overexpression ofPEP carboxylase leads to conversion of more phosphoenolpyruvate (PEP)into OAA, thus reducing the flux from PEP into pyruvate and subsequentlyinto acetyl-CoA. This leads to increased flux into the TCA cycle andthus into the pathway. Further, this overexpression also decreases theintracellular acetyl-CoA pools available for the ethanol-forming enzymesto work with, thus reducing the formation of ethanol and acetate. Theincreased flux towards oxaloacetate also helps reduce pyruvate andalanine secretion. Additionally, increased availability of oxaloacetatecan reduce the cellular needs for respiration and also the amount ofcarbon lost as CO₂. The gene ppc was cloned on the plasmid pZS*-13S asdescribed above.

Table 51 shows the BDO production in 96-well plates from the strain 3162from 4 replicates. The gene ppc (#525) was overexpressed on pZS*with apA promoter as described before and an RBK variant 5K was created. Hoststrain 3162 is based on strain ECKh-432 whose construction is describedabove and in US20110045575. Notable modifications include deletions inadhE, ldhA, pflB, mdh, and arcA. Strain ECKh-432 also containschromosomally integrated copies of sucD from Porphyromonas gingivalis(encoding CoA-dependant succinate semialdehyde dehydrogenase), sucA fromMycobacterium bovis (encoding alpha-ketoglutarate decarboxylase), 4hbdfrom P. gingivalis (encoding 4-hydroxybutyrate dehydrogenase), and 4hbdfrom Clostridium kluyveri (encoding 4-hydroxybutyrate dehydrogenase).Strain 3162 contains deletions in the native alcohol dehydrogenase genesadhP, yqhD, yahK, and yjgB. Additionally, strain 3162 contains achromosomally integrated copy of 4-hydroxybutyryl-CoA transferaseexpressed from the pA promoter at the attB locus. It also has deletionsof cytochrome oxidases, cyoABCD and appBC. The aldehyde dehydrogenaseand alcohol dehydrogenase genes for converting 4-hydroxybutyryl-CoA into4-hydroxybutyraldehyde and subsequently to 1,4-butanediol (BDO) areexpressed on the pPSX23R plasmid. The gene candidates for these enzymesteps have already been described. FIG. 71 shows the average BDO and 4HBfor the culture and compares it to 4 replicates from the control strain3162. The same comparison is made in FIG. 72 for the ethanol numbers.Higher BDO and 4HB were observed in cells with ppc overexpressed. Asexpected, lower ethanol numbers were seen.

TABLE 51 BDO, 4-hydroxybutyrate (4HB) and ethanol data for four4HB-producing replicates that had the gene ppc overexpressed on pZS*after 24 hours of culture time in 96-well plates. The top 4 rows showthe replicate data for the control cultures. All the concentrations arein mM. Host Plasmid #1 OD 4HB BDO EtOH 3162 pZS*13S 3.08 5.16 23.3 2.113162 pZS*13S 2.96 5.23 24.5 2.27 3162 pZS*13S 2.76 4.98 25.2 2.23 3162pZS*13S 3.05 6.02 26 2.3 3162 pZS*13S-(5k)525 3.02 9.18 29.9 1.59 3162pZS*13S-(5k)525 2.50 9.32 28.8 1.47 3162 pZS*13S-(5k)525 3.24 9.3 281.49 3162 pZS*13S-(5k)525 3.38 24.5 0.72 0.69

EXAMPLE XXXII Overexpression of Alpha-Ketoglutarate Dehydrogenase

This example describes overexpression of alpha-ketoglutaratedehydrogenase.

This enzyme complex is formed by sucA, sucB and lpdA in Escherichiacoli. It converts alpha-ketoglutarate into succinyl-CoA that issubsequently channeled into the BDO pathway. A limitation in thispathway, including any limitation in the capacity of alpha-ketoglutaratedehydrogenase, can lead to the formation of glutamate via, for example,glutamate dehydrogenase (gdhA). This leads to a carbon loss andreduction in yield. Expressing an extra copy of sucAB and lpdA in theBDO-producing strain helped reduce glutamate by significant levels. Itcan also pull flux through the TCA cycle and help reduce other C2(ethanol and acetate) and C3 byproducts (alanine, pyruvate). sucAB andlpdA were cloned on pZS*plasmid under the pA promoter as describedabove. ALD and ADH required for BDO production were expressed on pPSX23Ras described earlier.

Table 52 shows the 4HB and BDO production in 96-well plates from 4replicates of strain 3933 after 24 hours of culture time. Host strain3933 is based on strain ECKh-432 whose construction is described aboveand in US20110045575. Notable modifications include deletions in adhE,ldhA, pflB, mdh, and arcA. Strain ECKh-432 also contains chromosomallyintegrated copies of sucD from Porphyromonas gingivalis (encodingCoA-dependant succinate semialdehyde dehydrogenase), sucA fromMycobacterium bovis (encoding alpha-ketoglutarate decarboxylase), 4hbdfrom P. gingivalis (encoding 4-hydroxybutyrate dehydrogenase), and 4hbdfrom Clostridium kluyveri (encoding 4-hydroxybutyrate dehydrogenase). Itcontains deletions in the native alcohol dehydrogenase genes adhP, yqhD,yahK, and yjgB. The strain contains a chromosomally integrated copy of4-hydroxybutyryl-CoA transferase expressed from the pA promoter at theattB locus. It also has deletions of cytochrome oxidases, cyoABCD andappBC, and adh is integrated on the chromosome under the promoter p119at the hemN locus.

TABLE 52 BDO, 4HB and ethanol data for four 4HB producing replicates ofstrain 3933 that had the genes sucAB-lpdA overexpressed on a plasmid.The bottom 4 rows show the replicate data for the control cultures ofthe host strain 3933. All the concentrations are in mM and were measuredafter 24 hours of culture time in 96-well plates. 1438 refers to thenative sucA gene (b0726), 1439 refers to the native wild type sucB gene(b0727), the last gene in the biobrick on pZS* is a mutant version oflpdA (b0116) described previously in Example XIV. This gene is a subunitof the pyruvate dehydrogenase complex in Escherichia coli and has beenmodified such that pyruvate dehydrogenase is not inhibited by NADH. Themutations were made as described in Yim et al., Nature Chemical Biology7:445-452 (2011)). Host plasmid_1 OD 4HB BDO Glutamate 3933pZS*13S_1438-1439-160D345K-lpdA-KP 2.91 6.27 35.00 0.03 3933pZS*13S_1438-1439-160D345K-lpdA-KP 2.89 6.37 33.50 0.03 3933pZS*13S_1438-1439-160D345K-lpdA-KP 2.94 6.52 32.40 0.04 3933pZS*13S_1438-1439-160D345K-lpdA-KP 2.96 5.76 31.10 0.04 3933 pZS*13S3.06 6.44 38.30 2.68 3933 pZS*13S 3.05 5.42 36.40 2.20 3933 pZS*13S 3.225.36 36.40 2.21 3933 pZS*13S 3.12 4.95 35.00 2.08

FIG. 73 shows the average BDO and 4HB numbers from four replicates of a4HB producing host strain that had the genes sucAB and mutant lpdAoverexpressed on pZS*compared with the corresponding averages from fourreplicates of the control strain. ALD and ADH were expressed on theplasmid pPZSX23R. FIG. 74 shows the reduction in the average glutamatenumbers from the four replicates of the strain that had the genes sucABand mutant lpdA overexpressed on pZS*and compared with those from thecontrol. These results show that expression of sucAB resulted in adramatic reduction in the amount of glutamate produced and did notadversely affect the production of BDO or 4HB.

EXAMPLE XXXIII Overexpression of a Non-Phosphotransferase Sugar UptakeSystem

This example describes overexpression of glucokinase, galP and or glf asrepresentatives of a non-phosphotransferase (PTS) sugar uptake system.

The primary mode of glucose uptake in the host strains is the PTSsystem. Every molecule of glucose converted into glucose-6-phosphate(G6P) via this system is accompanied by the conversion of one moleculeof PEP into pyruvate. Note that PEP is converted into oxaloacetate viaPEP carboxylase in the strains. Additionally, PEP is also converted intopyruvate (by the PTS system as well as pyruvate kinase) and subsequentlyinto acetyl-CoA by pyruvate dehydrogenase in the strains. The BDOpathway requires equimolar amounts of oxaloacetate and acetyl-CoA forforming each mole of BDO. The fixed stoichiometry associated with thePTS system disturbs this balance, leading to higher ratios of acetyl-CoArelative to oxaloacetate. This then leads to production of ethanol andacetate in the cells. The overexpression of non-PTS mode of sugar uptakehelps alleviate this issue and balance the amount of oxaloacetateavailable in comparison to acetyl-CoA, thus increasing flux through theBDO pathway, reducing the C2 byproducts, acetate and ethanol, andreducing the C3 byproducts, pyruvate and alanine.

The primary mechanism of non-PTS glucose uptake is via a permease suchas galP to import the sugar and then its ATP-dependent phosphorylationvia the kinase encoded by glk. An alternate way to uptake glucose is viathe glucose facilitator, glf. While E. coli does not have thisfacilitator, one was cloned from Zymomonas mobilis and introduced intoE. coli (Parker et al. Mol. Microbiol. 15(5):795-802 (1995)).

EXAMPLE XXXIV Expression of Gamma-butyrolactone Esterase

This example describes expression of a gamma-butyrolactone esterase.

Gamma-butyrolactone (GBL) is a byproduct formed during the fermentationof sugars to 1,4-butanediol (BDO). It is formed from the unstablepathway intermediate 4-hydroxybutyryl-CoA. To a small extent, it canalso formed by spontaneous lactonization of 4-hydroxybutyrate.

Hydroxyacids and their corresponding lactones exist in pH-dependentequilibrium with each other. Lactonization is acid-catalyzed andfavorable at low pH. Under alkaline conditions the equilibrium is drivento the open-chain hydroxycarboxylate anion. At the cytoplasmic pH,typically 7.4, hydrolysis of the GBL lactone is favored (FIG. 75). Thehydrolysis of GBL to 4-HB can be accelerated in the presence of anenzyme with GBL esterase activity, thus improving the yield andeliminating a byproduct that can complicate downstream separations ofthe product.

Esterases from Yersinia intermedia 29909 and Agrobacterium tumefaciensstr. C58 (Carlier et al., Mol. Plant. Microbe Interact. 17(9):951-957(2004)) were identified and selected as exemplary esterase genes. Thenucleotide sequence of the Yersinia gene (locus yinte0001 13710) isshown in FIG. 89. The nucleotide sequence of the gene from Agrobacteriumtumefaciens is shown in FIG. 90.

Table 53 shows BDO, 4HB and GBL data from 96 well-plates of fourreplicates of a strain that had the esterase integrated (Strain 2387)and compared with the control with no esterase (strain 2237). Hoststrain 2237 is based on strain ECKh-432 whose construction is describedabove and in US20110045575. Notable modifications include deletions inadhE, ldhA, pflB, mdh, and arcA. Strain ECKh-432 also containschromosomally integrated copies of sucD from Porphyromonas gingivalis(encoding CoA-dependant succinate semialdehyde dehydrogenase), sucA fromMycobacterium bovis (encoding alpha-ketoglutarate decarboxylase), 4hbdfrom P. gingivalis (encoding 4-hydroxybutyrate dehydrogenase), and 4hbdfrom Clostridium kluyveri (encoding 4-hydroxybutyrate dehydrogenase).Strain 2237 contains deletions in the native alcohol dehydrogenase genesadhP, yqhD, yahK, and yjgB. It also contains a chromosomally integratedcopy of 4-hydroxybutyryl-CoA transferase expressed from the pA promoterat the attB locus. An additional deletion in poxB was also introduced.

TABLE 53 BDO, 4HB and GBL data from 96 well-plates of four replicates ofa strain that had the esterase integrated (Strain 2387) and comparedwith the control with no esterase (strain 2237). All concentrations arein mM and were measured after 19 hours of culture time. Host OD 4HB BDOGBL 2237 3.56 2.50 27.70 4.54 2237 3.43 2.54 32.20 5.00 2237 3.37 2.4427.10 4.34 2237 3.54 2.19 22.60 3.26 2387 3.80 0.00 23.60 0.00 2387 3.872.59 24.70 0.65 2387 4.00 2.62 27.70 0.59 2387 4.30 3.20 29.00 0.68

FIG. 76 shows the average BDO and 4HB numbers from four replicates of ahost strain capable of producing 4-hydrobutyryl-CoA that had theesterase integrated (2387) compared with the corresponding averages fromfour replicates of the control strain (2237). FIG. 77 shows the averageGBL numbers from four replicates of a host strain that had the esteraseintegrated (2387) compared with the corresponding averages from fourreplicates of the control strain (2237). Lower GBL was observed with thestrains that had the esterase integrated. These results demonstrateexpression of an enzyme having gamma-butyrolactone esterase activitywithout adversely affecting production of BDO or 4HB.

EXAMPLE XXXV Deletion of Succinyl-CoA Synthetase

This example describes deletion of succinyl-CoA synthetase.

Succinyl-CoA synthetase is encoded by sucCD in Escherichia coli. Thedeletion of arcA highly upregulates the expression of these genes (andthe entire sdh-suc operon) in the cells. Repeated rounds of flux throughthe TCA cycle leads to carbon loss as CO₂. The deletion of sucCD blockedthe TCA cycle downstream of succinyl-CoA, reducing the CO₂ losses by˜70% and leading to a corresponding increase in BDO titers and yields.

The deletion of sucCD was introduced in host strain 4070. This strain isbased on strain ECKh-432 whose construction is described above and inUS20110045575. Notable modifications include deletions in adhE, ldhA,pflB, mdh, and arcA. Strain ECKh-432 also contains chromosomallyintegrated copies of sucD from Porphyromonas gingivalis (encodingCoA-dependant succinate semialdehyde dehydrogenase), sucA fromMycobacterium bovis (encoding alpha-ketoglutarate decarboxylase), 4hbdfrom P. gingivalis (encoding 4-hydroxybutyrate dehydrogenase), and 4hbdfrom Clostridium kluyveri (encoding 4-hydroxybutyrate dehydrogenase).Strain 4070 contains deletions in the native alcohol dehydrogenase genesadhP, yqhD, yahK, and yjgB. Additionally, this strain contains achromosomally integrated copy of 4-hydroxybutyryl-CoA transferaseexpressed from the p119 promoter at the attB locus. It also hasdeletions of cytochrome oxidases, cyoABCD and appBC. The adh wasintegrated on the chromosome under the promoter p119 at the hemN locus.

TABLE 54 BDO and 4HB data from 96 well-plates of four replicates of a4-HB producing strain that had the sucCD genes deleted (Strain 4269)compared with the no deletion control (strain 4070). Higher 4HB wasobserved with strains that had the sucCD deleted. All concentrations arein mM and were measured after 24 hours of culture time. Host OD 4HB BDO4070 3.62 0.289 37.2 4070 3.58 0.777 41.4 4070 3.33 0.377 28.5 4070 3.750.792 54.4 4269 2.95 8.93 50.5 4269 2.68 8.07 50.5 4269 3.34 8.65 50.14269 2.94 8.55 51.8 Host plasmid_1 OD 4HB BDO 4070 pZS*13S-p100-ALD 3.620.289 37.2 4070 pZS*3S-p100-ALD 3.58 0.777 41.4 4070 pZS*13S-p100-ALD3.33 0.377 28.5 4070 pZS*13S-p100-ALD 3.75 0.792 54.4 4269pZS*13S-p100-ALD 2.95 8.93 50.5 4269 pZS*13S-p100-ALD 2.68 8.07 50.54269 pZS*13S-p100-ALD 3.34 8.65 50.1 4269 pZS*13S-p100-ALD 2.94 8.5551.8

FIG. 78 shows the average BDO and 4HB numbers from four replicates of ahost strain that had the genes sucCD deleted (4269) compared with thecorresponding averages from four replicates of the control strain(4070). These results demonstrate increased yield of BDO and 4HB withthe deletion of sucCD.

EXAMPLE XXXVI Deletion of Acyl CoenzymeA Thioesterases

This example describes deletion of acyl coenzymeA thioesterases ybgC andtesB.

One of the intermediates in the BDO pathway is 4-hydroxybutyryl-CoA.This compound can spontaneously or enzymatically get cyclized to formGBL (gamma-butyryl-lactone). E. coli possesses some reversible CoAthioesterases reported to have broad substrate specificity on CoAsubstrates of C4 to C6 groups. The deletion of these gene candidateseliminated approximately 50% of the GBL formation via the pathway. Suchactivities can be found analogously in other organisms.

The deletions of tesB and ybgC were made in host strain 1136. Thisstrain is based on strain ECKh-432 whose construction is described aboveand in US20110045575. Notable modifications include deletions in adhE,ldhA, pflB, mdh, and arcA. Strain ECKh-432 also contains chromosomallyintegrated copies of sucD from Porphyromonas gingivalis (encodingCoA-dependant succinate semialdehyde dehydrogenase), sucA fromMycobacterium bovis (encoding alpha-ketoglutarate decarboxylase), 4hbdfrom P. gingivalis (encoding 4-hydroxybutyrate dehydrogenase), and 4hbdfrom Clostridium kluyveri (encoding 4-hydroxybutyrate dehydrogenase).Strain 1136 has additional deletions in ndh (NADH dehydrogenase II) andsuccinate semialdehyde dehydrogenase encoding genes, sad and gabD.

TABLE 55 OD, BDO, 4HB and GBL numbers of four replicates of a straincapable of producing 4-hydroxybutyryl-CoA that had both tesB and ybgCdeleted (1197) and compared with the control with neither of thesedeletions (Host 1136). All concentrations are in mM and were measuredafter 40 hours of culture time in 20 mL bottles. Host OD BDO 4HB GBL1136 2.428 26.2 3.1 1.8 2.344 27.4 2.7 1.7 2.392 26.6 3.3 1.8 1197 3.32429.9 2.8 1.0 2.712 29.1 2.6 0.9 2.924 32.0 2.9 0.9

FIG. 79 shows the average BDO and 4HB numbers from three replicates of ahost strain that had the genes ybgC and tesB deleted (1197) comparedwith the corresponding averages from three replicates of the controlstrain (1136). FIG. 80 shows the average GBL numbers from threereplicates of a host strain that had the genes ybgC and tesB deleted(1197) compared with the corresponding averages from three replicates ofthe control strain (1136). These results demonstrate that deletion ofone or more acyl coenzyme A thioesterases decreased production ofgamma-butyrolactone, increased production of BDO and had no adverseaffect on production of 4HB.

EXAMPLE XXXVII Deletions to Prevent or Reduce Backflux into the Pathway

This example describes deletions to prevent backflux into the pathway.

At high titers of 1,4-butanediol (BDO) in the culture, it starts to getreimported into the cells. Once inside the cells, it is metabolizedthrough the TCA cycle. To prevent this backflux of BDO into the cells,alcohol dehydrogenases that were identified as candidates for thereverse flux were deleted. The candidates were identified by twoapproaches, first, all the alcohol dehydrogenases were listed andprioritized based on their substrate specificity. Microarrays wereconducted to determine how high each one of them was expressed. Based onthis, a short list of 5 candidates was generated from a list ofapproximately 35 adh candidates. Secondly, protein fractions withendogenous backflux activity (NADP+ dependent BDO conversion to4-hydroxybutyraldehyde) were isolated from strain 1427 (alcoholdehydrogenase encoded by yqhD removed and no downstream pathway present,cells accumulate 4HB but do not make BDO) grown microaerobically in MM9media. The sample was purified while following the backflux activity.The strategy employed is outlined below.

Based on early experiments, the endogenous ADH activity was found to beprimarily NADPH dependent and salted out in high (>35%) ammoniumsulfate. A fresh sample of 1427 cells from a 2 L LB preparation grownaerobically overnight was lysed using the microfluidizer at 15,000 psi.The sample was then fractionated via ammonium sulfate. The 35% pelletcontained little ADH activity and was not processed further, while the60% pellet contained active ADH with significant backflux activity andwas moved onto further purification.

Anion Exchange Purification. The 60% ammonium sulfate pellet wasresuspended in a low salt buffer and dialyzed overnight to remove theresidual ammonium sulfate. The sample was loaded onto a 5 ml Q-sepharosecolumn (strong anion exchange) and eluted via a slow increasing saltgradient. The profile for the anion exchange elution, backflux activity,as well as the SDS page for the fractions is shown below in FIG. 81.

The first large peak shown on the left is off-scale and corresponds toproteins that did not bind the column. The peak had low backfluxactivity. The next peak shown in the middle of the chromatogram hadsignificant backflux activity. An SDS-PAGE of this peak was run andindicates that further purification is needed in order to isolate theprotein of interest.

Size Exclusion Chromatography. The peak backflux activity from anionexchange was pooled and concentrated to allow for application to a S200sephacryl size exclusion column. The column successfully removedimpurities from the sample and the results are shown below (FIG. 82).

The results of the sizing column indicated that the protein of interestbehaved as a mid-range MW protein, ˜50 kDa. However, it was not clearwhat the identity of the protein was, but it narrowed down toapproximately 20 bands on the gel. Additional chromatography wasperformed to attempt to clean up the sample prior to mass spec analysis.

Hydrophobic Interaction Chromatography.

The peak with the highest backflux activity from the size exclusioncolumn was pooled and loaded on a phenyl sepharose column (hydrophobicinteraction column). The ionic strength of the sample was increased tofacilitate binding to the resin. The results are shown below (FIG. 83).

The sample shown on the SDS-PAGE was analyzed by mass spectrometry inorder to identify the protein(s) that is responsible for the backflux.From the gel there are 5 prominent bands alongside a small handful ofbackground proteins (˜5).

The purified sample was analyzed by mass spectrometry analysis, and alist of 30 proteins was obtained. From this list the gene selected was:

Accession Name Gene gi|170083708 Zn-dependent/NAD(P)- yjgB bindingalcohol dehydrogenase

The yjgB gene was cloned with a streptavidin tag, expressed and purifiedin order to characterize its properties. The results of thepurification, as analyzed by SDS-PAGE, are shown in FIG. 84. The proteinexpressed well, and acceptable yields of purified protein were obtained.Initial characterization with BDO as a substrate was performed in thepresence of NADP+ and robust activity was confirmed (see FIG. 85).

On the basis of the protein fractionation and the microarray results,four genes were selected and deleted on top of each other to obtainstrain 1889 from 1872. The genes were yqhD (b3011; GenBank NP_417484.1,GI:16130909), yjgB (b4269; GenBank NP_418690.4, GI:90111716), yahK(b0325; GenBank NP_414859.1, GI:16128310) and adhP (b1478; GenBankNP_415995.4, GI:90111280).

The deletions of these four alcohol dehydrogenases were introduced instrain 1872. This strain is based on strain ECKh-432 whose constructionis described above and in US20110045575. Notable modifications includedeletions in adhE, ldhA, pflB, mdh, and arcA. Strain ECKh-432 alsocontains chromosomally integrated copies of sucD from Porphyromonasgingivalis (encoding CoA-dependant succinate semialdehydedehydrogenase), sucA from Mycobacterium bovis (encodingalpha-ketoglutarate decarboxylase), 4hbd from P. gingivalis (encoding4-hydroxybutyrate dehydrogenase), and 4hbd from Clostridium kluyveri(encoding 4-hydroxybutyrate dehydrogenase). Strain 1872 contains achromosomally integrated copy of 4-hydroxybutyryl-CoA transferaseexpressed from the pA promoter at the attB locus.

TABLE 56 OD, BDO, 4HB and GBL numbers of four replicates of a strainthat had all four alcohol dehydrogenases identified for carryingbackflux deleted (1889) compared with the control with neither of thesedeletions (Host 1872). The gene cat2 for 4HB conversion into4-hydroxybutyryl-CoA was expressed on F′ whereas ALD and ADH wereexpressed on pZS*-13S as described already. All concentrations are in mMand were measured after 24 hours of culture time in 96 well-plates. HostOD 4HB BDO GBL 1872 4.61 3.3 60.8 12.5 1872 6.09 3.6 90.2 15.3 1872 5.883.1 84.4 17.3 1872 5.84 2.9 96.3 15.9 1889 5.94 3.6 92.6 14.0 1889 6.593.3 88.9 13.1 1889 6.59 3.3 82.4 13.4 1889 6.58 3.6 89.4 14.4

FIG. 86 shows the average BDO, 4HB and GBL numbers for the strain 1872versus strain 1889. These results demonstrate that metabolicmodifications that decrease backflux result in an increased yield of BDOand no adverse affect on production of 4HB.

EXAMPLE XXXVIII Deletions to Improve the Energetic Efficiency ofOxidative Phosphorylation

This example describes deletions to improve the energetic efficiency ofoxidative phosphorylation.

The electron transport chain of Escherichia coli has multiple NADHdehydrogenases and cytochrome oxidases, with varying ability totranslocate protons. For example, NADH dehydrogenase II in E. coli is anNADH consuming system that is not linked with proton translocation(H+/2e−=0) whereas NADH dehydrogenase I encoded by nuo is reported totranslocate 4 protons per pair of electrons. The major role of Ndh-II isto oxidize NADH and to feed electrons into the respiratory chain (Yun etal., J. Appl. Microbiol. 99:1404-1412 (2005)). The affinity of NdhII forNADH is relatively low (Hayashi et al., Biochim. Biophys. Acta 977:62-69(1989)). It has been suggested that NdhII may operate to regulate theNADH pool independently of energy generation and is likely to beimportant when the capacity of bacteria to generate energy exceedsdemand. The ndh gene has been shown to be repressed by the fnr geneproduct in such a way that the expression is optimal under conditions ofhigh oxygen concentrations. The deletion of ndh would thus help inimproving the energy efficiency of the cell. Similarly, there areseveral other NADH dehydrogenases that are not known to translocate anyprotons and thus do not help in ATP production, for example, wrbA, yieF,and kefF in E. coli. Homologues of these can be found in other organismsand eliminated to improve the ATP production for every unit of oxygenconsumed.

Host strain 879 (the parent strain for ndh deletion) was based on strainECKh-432 whose construction is described above and in US20110045575.Notable modifications include deletions in adhE, ldhA, pflB, mdh, andarcA. Strain ECKh-432 also contains chromosomally integrated copies ofsucD from Porphyromonas gingivalis (encoding CoA-dependant succinatesemialdehyde dehydrogenase), sucA from Mycobacterium bovis (encodingalpha-ketoglutarate decarboxylase), 4hbd from P. gingivalis (encoding4-hydroxybutyrate dehydrogenase), and 4hbd from Clostridium kluyveri(encoding 4-hydroxybutyrate dehydrogenase). Strain 879 also hasdeletions of the succinate semialdehyde dehydrogenase genes, sad andgabD.

TABLE 57 The BDO, 4HB and GBL production in three replicates of thestrain that had ndh deleted compared with the replicates of the hoststrain (879) with ndh intact. The experiments were conducted in 20 mLbottles as described in the protocol. The ALD and ADH were present onthe plasmid pZS* to allow the conversion of 4-HB CoA to 4-HB aldehydeand subsequently to BDO. All concentrations are in mM and were measuredafter 24 hours of culture time. Host OD BDO GBL 4HB 879 1.61 3.27 0.831.28 879 2.45 4.15 0.99 1.14 956 2.31 4.48 0.63 1.01 956 2.01 4.31 0.590.96 956 2.54 4.92 0.59 1.00

FIG. 87 shows the average BDO, 4HB and GBL numbers for the strain 956versus strain 879. These results demonstrate that deletions that improvethe energy efficiency of oxidative phosphorylation increase the yield ofBDO and have no adverse affect on 4HB production.

EXAMPLE XXXIX Deletion of Cytochrome Oxidases

This example describes deletion of cytochrome oxidases.

On the electron output side of the electron transport chain, multiplecytochrome oxidases are present that have different energy-conservingefficiencies. The cytochrome bo complex, encoded by the cyo operon,actively pumps electrons over the membrane and results in an H+/2e−stoichiometry of 4. The cytochrome bd-I complex does not actively pumpprotons, but due to the oxidation of the quinol on the periplasmic sideof the membrane and subsequent uptake of protons from the cytoplasmicside of the membrane, which are used in the formation of water, the netelectron transfer results in a H+/2e− stoichiometry of 2. This isencoded by the cyd operon. Until recently, the proton translocationstoichiometry of cytochrome bd-II oxidase, encoded by appBC, was notknown, but it has now been established that this oxidase is electrogenic(Borisov et. al., Proc. Natl. Acad. Sci. USA 108:17320-17324 (2011)).These genes are normally induced upon entry into stationary phase orunder conditions of carbon and phosphate starvation (Atlung et al., J.Bacteriol. 179:2141-2146 (1997)).

Properties and abundance of the cytochrome oxidases: The cyd operon isregulated in an oxygen-dependent manner by fnr and arcA and theexpression is optimal under microaerophilic conditions (Calhoun et al.,J. Bacteriol. 175:3020-3025 (1993)). The bo-type oxidase predominateswhen E. coli is grown at high oxygen tension. The cyo operon is alsoregulated by both arcA and fnr. A literature study indicates that inaerobically grown cells the cytochrome o oxidase is present at a levelof approximately 304 molecules per cell and the cytochrome d oxidase at204 molecules per cell (Minohara et al., J. Biosci. Bioengineer.93(5):464-469 (2002)). Under anaerobic conditions, cytochrome d oxidaseis predominant and is present at about 606 molecules per cell incontrast to only 2 molecules per cell of cytochrome o oxidase. The Kmfor O₂ of cytochrome bd oxidase is much lower than that of thecytochrome bo oxidase (Minohara et al, supra, 2002). The former has a Kmof 10 nM, and cytochrome bo oxidase has a Km of ˜1 μM.

TABLE 58 Enzymatic characteristics of the cytochrome-bd oxidases from E.coli (From Bekker et al., supra, 2009). K_(m) In vivo sp V_(max) (mol(UQ-H₂) Cellular act (mol O/mol K_(m) (O₂) at content O₂/mol cytochromeat pH 7 pH 7 (nmol/g cytochrome Cytochrome bd/s) (μM) (μM) protein)bd/s) Cytochrome 218 ± 20 0.3^(h) 85 ± 5 90 ± 29 58 ± 11 bd-I Cytochrome818 ± 75 2.0 ± 0.3 250 ± 45 91 ± 32 70 ± 12 bd-II Cytochrome 225^(c)6.0^(d) 47^(e) ND ND bo

In a study attempting to determine the P/O ratios in aerobically-grownE. coli (Noguchi et al., J. Biochem. 136:509-515 (2004)), it wasdetermined that NDH-1 formed the predominant NADH dehydrogenase underboth standard feed and limiting feed of glucose. However, cytochrome bdoxidase was found to be present in higher concentrations than cytochromebo oxidase. The following table illustrates their findings on variousNADH dehydrogenases and cytochrome oxidases.

TABLE 59 Concentrations of NADH dehydrogenases and cytochrome oxidaseunder aerobic growth conditions with different glucose feedingstrategies (from Noguchi et al., supra, 2004) NDH-1 NDH-2 bo-typebd-type Conditions (Phase) (μmol/s/mg of protein) NDH-1/Total (%)bo-type/Total (%) Standard glucose (exponential: 3 h) 1.58 ± 0.07 1.11 ±0.06 1.38 ± 0.12 1.01 ± 0.10 58.8 ± 2.4 57.9 ± 4.5 (exponential: 5 h)1.35 ± 0.03 0.86 ± 0.03 0.70 ± 0.05 1.07 ± 0.04 61.0 ± 1.4 39.6 ± 2.6(early stationary: 8 h) 1.17 ± 0.01 0.88 ± 0.01 0.46 ± 0.03 0.98 ± 0.0257.1 ± 0.5 32.0 ± 1.9 Limited glucose (early stationary) 0.97 ± 0.050.70 ± 0.03 0.42 ± 0.04 0.75 ± 0.05  58.1 ± 2.29 36.0 ± 3.7

Effects of deleting cytochrome oxidases: A chemostat study has beenreported for understanding the ratios of specific oxygen consumptionrates for strains lacking different components of the electron transportchain between dilution rates of 0.1 and 0.7/hr (Calhoun et al., supra,1993). Strains that were constrained to use the bd-type oxidase hadincreased oxygen consumption rates as compared to those that used onlythe cytochrome bo oxidase by a factor of 1.45-1.13. The ratio of theoxygen consumption rates for a strain that had both ndh and cyo deletedas compared to the strain with only ndh deleted was between 1.46 and1.39. The authors of Calhoun et al., supra, 1993, concluded that thebd-type oxidase is less efficient than the bo-type oxidase but is stillcoupled.

In yet another study, it has been reported that E. coli wild-type cellscontaining both cytochrome bo and bd type terminal oxidases, pumpedprotons with a H+/O ratio of 4.5-4.9, but mutants with cytochrome booxidase deleted, showed ratios of 3.5-4.1 and the mutants withcytochrome bd oxidase showed a ratio of 4.8-5.6 (Minohara et al., supra,2002). Mutants which lacked both cyo and cyd operon could not grow underaerobic conditions, but those that overexpressed cytochrome bo oxidaseand those that overexpressed cytochrome bd oxidase grew as well as theparental W3110 strains. The cell yield of each of these strains was inproportion to their H+/O ratios. Interestingly, the strain which hadcydAB deleted showed very poor cell yield initially. The cell yieldswere, however, improved dramatically once the cytochrome bo oxidase wasoverexpressed. The authors Minohara et al., supra, 2002) hadhypothesized that the lower cell yields in the cyd mutant strain may becaused by the high H+ permeability of the membrane in the cyd mutant.They believed that the amounts of cytochrome bo in the mutant cellslacking the high-affinity oxygen-reducing cytochrome bd may not besufficient to reduce the oxygen molecules at a low concentration, andthus the cell membranes were injured by reactive oxygen species and weremade partially permeable. This cell stress could be relieved byoverexpressing cytochrome bo oxidase (Minohara et al., supra, 2002).

TABLE 60 Properties of various E. coli mutants grown in aglucose-limited chemostat culture at a dilution rate of 0.15/hr. Thecytochrome shown in parentheses is the active cytochrome oxidase. Eachof these strains also had nuo deleted (from Minohara et al., supra,2002). q_(glucose) Y_(glucose) q_(acetate) q_(ATP) q_(O2) {mmol {mmol (g[dry wt]) {mmol {mmol (g [dry wt])⁻¹ (g [dry wt])⁻¹ (g (g [dry wt])⁻¹ (g[dry wt])⁻¹ Mutant h⁻¹} h⁻¹} Glc)⁻¹ h⁻¹} h⁻¹} H⁺/2e⁻ MB30 (cytochromebd-I) 8.8 ± 1.3 3.2 0.26 1.5 ± 0.5 16.3 ± 2.2 2 MB37 (cytochrome bd-II)11.1 ± 0.6  6.0 0.14 5.7 ± 0.7 16.6 0.2 ± 0.1 MB34 (cytochrome bo) 6.4 ±0.4 2.3 0.36 0.4 ± 0.4 17.0 ± 0.4 4

A recent paper (Bekker et al., J. Bacteriol. 191:5510-5517 (2009))investigated the effects of deleting cytochrome bd-II oxidase. Thephenotype of the mutant strain lacking the cytochrome bd-II oxidaseincluded a smaller ubiquinone pool when grown under glucose-excessconditions. This strain also had a decreased oxygen flux inglucose-limited chemostat conditions. These results suggested thatcytochrome bd-II oxidase contributed significantly to the overallrespiratory electron flux. Wild type E. coli MG1655 cells had a specificrespiration rate that was approximately 46% higher than that of themutant strain that lacked appBC (8.7 vs. 5.9 mmol/gDCW.hr). These weremeasured in glucose-limited chemostats at a dilution rate of 0.2/hr. Inorder to improve the energetic efficiency of the cells, the appBC wasknocked out in the strains (Host strain 2424), and additionally cyoABCDwas knocked out (Strain 2471). The table below shows the data forproduction of the C4 metabolites in 96 well plates with each of thesestrains. An additional advantage of deleting the two cytochrome oxidases(encoded by appBC and cyoABCD) and ndh is that it makes the cellsrelatively robust to a range of oxygen transfer rates in biggerfermenters. Since cydAB has a fixed stoichiometry for protontranslacoation and so does nuo (NADH dehydrogenase I), the P/O ratio israther constant and so is the biomass rate in different parts of afermenter.

TABLE 61 The BDO, 4HB and GBL production in three replicates of thestrains 1889 and 2471. The ALD and ADH were present on the plasmid pZS*to allow the conversion of 4-HB CoA to 4-HB aldehyde and subsequently toBDO. All concentrations are in mM and were measured after 24 hours ofculture time in 96-well plates. Host OD 4HB BDO GBL 1889 3.63 5.71 57.204.48 1889 3.05 5.65 50.60 3.49 1889 3.40 4.14 51.90 3.90 1889 3.19 4.2353.10 3.69 2471 3.56 5.62 64.20 4.80 2471 2.96 5.73 53.30 2.75 2471 3.075.39 42.80 2.67 2471 3.11 4.01 45.90 2.66

FIG. 88 shows the average BDO, 4HB and GBL numbers of the strains, 1889(see above) and 2471. These results demonstrate that deletion ofcytochrome oxidases results in deletion of appBC resulted in a decreasein BDO and 4HB production, although both BDO and 4HB are still producedin good quantities, and these cells are expected to exhibit improvedenergy efficiency that may be advantageous in scaled up fermentationconditions.

The results disclosed herein demonstrate high yield of desired productssuch as 4-HB, 4-HBal, 4-HBCoA, BDO or putrescine, in particular BDO.Such modifications have been demonstrated to increase product yield ofBDO. Such beneficial modifications, including both increasing anddecreasing expression of enzymes, resulted in decreased CO₂ loss,decreased by-product formation such as ethanol, acetate, pyruvate,alanine, glutamate and GBL. Additional modifications decreased backfluxfrom desired products such as BDO into pathway intermediates orprecursors such as those in the TCA cycle. Additional improvements havebeen made by identifying enzymes that more efficiently carry out adesired pathway reaction and optionally subjecting the enzyme toevolution to generate variants having improved characteristics such asincreased substrate to product conversion, stability, and the like. Suchenzymes have included cat2, aldehyde dehydrogenase and aldehydedehydrogenase, as disclosed herein. In addition, various promoters havebeen tested to identify promoter variants that provide more efficienttranscription of a gene. Additionally, as disclosed herein, variousbackflux and by-product reactions can decrease the efficiency of productformation in a desired pathway. In some cases, product yield can beincreased by decreasing expression of a pathway enzyme if higherexpression of the pathway enzyme results in an increase in by-productformation. For example, such a result has been observed with aldehydedehydrogenas, in which lowere expression reduces ethanol productionwithout lowering BDO production.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties, includingGenBank and GI number publications, are hereby incorporated by referencein this application in order to more fully describe the state of the artto which this invention pertains. Although the invention has beendescribed with reference to the examples provided above, it should beunderstood that various modifications can be made without departing fromthe spirit of the invention.

What is claimed is:
 1. A non-naturally occurring microbial organism,said microbial organism having a 4-hydroxybutyrate pathway and beingcapable of producing 4-hydroxybutyrate, wherein said microbial organismcomprises genetic modifications, said genetic modifications comprising:(A) a genetic modification that increases expression ofphosphoenolpyruvate carboxylase; (B) a genetic modification thatincreases expression of alpha-ketoglutarate dehydrogenase; and (C) agenetic modification that decreases expression of an acyl coenzyme Athioesterase.
 2. The microbial organism of claim 1, wherein: (K) themicrobial organism has decreased production of ethanol, acetate,pyruvate or alanine, or a combination thereof, relative to a parentmicrobial organism in the absence of said genetic modification; (L) themicrobial organism has decreased production of glutamate relative to aparent microbial organism in the absence of said genetic modification;and (M) the microbial organism has decreased production ofgamma-butyrolactone relative to a parent microbial organism in theabsence of said genetic modification.
 3. The microbial organism of claim1, wherein said microbial organism further comprises a4-hydroxybutyryl-CoA pathway.
 4. A method for producing4-hydroxybutyrate, comprising culturing the non-naturally occurringmicrobial organism of claim 1 under conditions and for a sufficientperiod of time to produce 4-hydroxybutyrate.
 5. The method of claim 4,wherein: (K) the microbial organism has decreased production of ethanol,acetate, pyruvate or alanine, or a combination thereof, relative to aparent microbial organism in the absence of said genetic modification;(L) the microbial organism has decreased production of glutamaterelative to a parent microbial organism in the absence of said geneticmodification; and (M) the microbial organism has decreased production ofgamma-butyrolactone relative to a parent microbial organism in theabsence of said genetic modification.
 6. The method of claim 4, whereinsaid non-naturally occurring microbial organism is in a substantiallyanaerobic culture medium.
 7. The microbial organism of claim 1, whereinsaid microbial organism further comprises a genetic modificationselected from: (D) a genetic modification that increases expression of anon-phosphotransferase (PTS) glucose uptake system; (E) a geneticmodification that increases expression of a gamma-butyrolactoneesterase; (F) a genetic modification that decreases expression ofsuccinyl-CoA synthetase; (G) a genetic modification that decreasesexpression of an alcohol dehydrogenase (H) a genetic modification thatdecreases expression of a non-energy-producing NADH dehydrogenase; (I) agenetic modification that decreases expression of a cytochrome oxidase;and (J) a combination of two or more of the genetic modifications ofparts (C)-(I).
 8. The microbial organism of claim 7, wherein: (N) themicrobial organism of part (D) has a genetic modification comprisingincreased expression of a permease, glucokinase, or a glucosefacilitator, or a combination thereof; (O) the microbial organism ofpart (E) has decreased production of gamma-butyrolactone relative to aparent microbial organism in the absence of said genetic modification;(P) the microbial organism of part (F) has increased production of4-hydroxybutyrate relative to a parent microbial organism in the absenceof said genetic modification; (Q) the microbial organism of part (G) hasdecreased backflux from a downstream product of the 4-hydroxybutyratepathway relative to a parent microbial organism in the absence of saidgenetic modification; (R) the microbial organism of part (H) hassuppressed depletion of the NADH pool or increased energy efficiency inthe microbial organism, or a combination thereof, relative to a parentmicrobial organism in the absence of said genetic modification; (S) themicrobial organism of part (I) has increased energy efficiency relativeto a parent microbial organism in the absence of said geneticmodification; or (T) the microbial organism of part (I) has increasedtolerance to a range of oxygen concentrations relative to a parentmicrobial organism in the absence of said genetic modification.
 9. Themethod of claim 4, wherein said microbial organism further comprises agenetic modification selected from: (C) a genetic modification thatincreases expression of a non-phosphotransferase (PTS) glucose uptakesystem; (D) a genetic modification that increases expression of agamma-butyrolactone esterase; (E) a genetic modification that decreasesexpression of succinyl-CoA synthetase; (F) a genetic modification thatdecreases expression of an alcohol dehydrogenase (G) a geneticmodification that decreases expression of a non-energy-producing NADHdehydrogenase; (H) a genetic modification that decreases expression of acytochrome oxidase; and (I) a combination of two or more of the geneticmodifications of parts (C)-(I).
 10. The method of claim 9, wherein: (N)the microbial organism of part (C) has a genetic modification comprisingincreased expression of a permease, glucokinase, or a glucosefacilitator, or a combination thereof; (O) the microbial organism ofpart (D) has decreased production of gamma-butyrolactone relative to aparent microbial organism in the absence of said genetic modification;(P) the microbial organism of part (E) has increased production of4-hydroxybutyrate relative to a parent microbial organism in the absenceof said genetic modification; (Q) the microbial organism of part (F) hasdecreased backflux from a downstream product of the 4-hydroxybutyratepathway relative to a parent microbial organism in the absence of saidgenetic modification; (R) the microbial organism of part (G) hassuppressed depletion of the NADH pool or increased energy efficiency inthe microbial organism, or a combination thereof, relative to a parentmicrobial organism in the absence of said genetic modification; (S) themicrobial organism of part (H) has increased energy efficiency relativeto a parent microbial organism in the absence of said geneticmodification; or (T) the microbial organism of part (H) has increasedtolerance to a range of oxygen concentrations relative to a parentmicrobial organism in the absence of said genetic modification.
 11. Themicrobial organism of claim 1, wherein the microbial organism has agenetic modification comprising at least two genetic modifications thatdecrease expression of at least two acyl coenzyme A thioesterases. 12.The method of claim 4, wherein the microbial organism has a geneticmodification comprising at least two genetic modifications that decreaseexpression of at least two acyl coenzyme A thioesterases.